Agricultural pheromone compositions comprising positional isomers

ABSTRACT

The present disclosure provides pheromone compositions. In some aspects, the compositions taught herein comprise a pheromone chemically corresponding to the pheromone naturally produced by a given insect, along with at least one positional isomer of said pheromone. In various aspects, pheromone compositions of the present disclosure are able to modulate the response of the insect based on the ratio of natural pheromone to its positional isomer.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S. Provisional Application No. 62/255,215, filed on Nov. 13, 2015, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: PRVI_010_01US_SeqList_ST25.txt, date created: Nov. 11, 2016, file size≈24 kilobytes).

BACKGROUND

Insects are estimated to cause global crop losses of approximately $250Bn—equivalent to 15% of global crop yield. Broad-spectrum insecticides, such as pyrethroid, organophosphate, and carbamate insecticide sprays, are currently used to combat such losses. However, these insecticides are harmful to both humans and the environment. In addition, the widespread use of insecticides has resulted in the evolution of resistant insects. For example, small-plot insecticide evaluations and scattered control failures in commercial sweet corn fields suggest that corn earworm populations in the Midwestern United States and southeastern Canada are gaining widespread resistance to pythrethroid-based insecticides. The rising frequency of resistant insects and the greater ease with which such insects migrate in a global economy have led to super-bugs that are causing multi-billion dollar losses. The cotton bollworm (Helicoverpa armigera) in Brazil and the corn rootworm (Diabrotica virgifera virgifera) in the United States are also contemporary illustrations of this trend. Furthermore, controlling infestations with broad-spectrum insecticides also reduces populations of beneficial insects, which leads to an outbreak of secondary pests, such as mites.

Thus, there exists a need for an insect management practice which prevents crop damage but does not have the harmful consequences of broad-spectrum insecticides.

BRIEF SUMMARY OF THE DISCLOSURE

In a first aspect, the disclosure provides for an insect pheromone composition for modifying the behavior of a target member of the order Lepidoptera. In some embodiments the pheromone composition comprises: (a) a first synthetically derived insect pheromone, having a chemical structure corresponding to that of a natural insect pheromone produced by a given target member of the order Lepidoptera; and (b) a positional isomer of said first synthetically derived insect pheromone, wherein said positional isomer is not naturally produced by the target member of the order Lepidoptera.

In some embodiments, the positional isomer is not produced by a member of the order Lepidoptera. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the family Noctuidae or Plutellidae. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the genus Helicoverpa, Plutella, Spodoptera, or Chrysodeixis. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Helicoverpa zea. In other embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Helicoverpa armigera. In still other embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Plutella xylostella. In yet other embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Spodoptera frugiperda. In other embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Chrysodeixis includens.

In some embodiments, the first synthetically derived insect pheromone is present in the composition in a ratio of from about 99% to about 1%, relative to the positional isomer, which is present in the composition in a ratio of from about 1% to about 99%. In other embodiments, the first synthetically derived insect pheromone is present in the composition in an amount of from about 99% to about 1% w/w. In yet other embodiments, the positional isomer is present in the composition in an amount of from about 99% to about 1% w/w.

In some embodiments, the first synthetically derived insect pheromone is Z-11-hexadecenal. In some embodiments, the first synthetically derived insect pheromone is Z-11-hexadecenal and the positional isomer is Z-5-hexadecenal.

In another aspect, the insect pheromone composition further comprising: (c) a second synthetically derived insect pheromone, having a chemical structure corresponding to that of a natural insect pheromone produced by a given target member of the order Lepidoptera; and (d) optionally, a positional isomer of said second synthetically derived insect pheromone, wherein said positional isomer is not naturally produced by the target member of the order Lepidoptera. In some embodiments, the second synthetically derived insect pheromone is Z-9-hexadecenal. In some embodiments, the second synthetically derived insect pheromone is Z-9-hexadecenal and the positional isomer of the second synthetically derived insect pheromone is present and is Z-7-hexadecenal. In some embodiments, the first synthetically derived insect pheromone is Z-11-hexadecenal and the positional isomer of the first synthetically derived insect pheromone is Z-5-hexadecenal, and wherein the second synthetically derived insect pheromone is Z-9-hexadecenal and the positional isomer of the second synthetically derived insect pheromone is present and is Z-7-hexadecenal.

In some embodiments, the pheromone composition further comprises at least one additional synthetically derived insect pheromone. In some embodiments, the insect pheromone composition further comprises an agriculturally acceptable adjuvant or carrier.

In some embodiments, an insect pheromone composition for modifying the behavior of male Helicoverpa sp., is disclosed herein which comprises: (a) Z-11-hexadecenal and Z-5-hexadecenal; and (b) an agriculturally acceptable adjuvant or carrier. In some such embodiments, the Z-11-hexadecenal is present in the composition in a ratio of from about 99% to about 1%, relative to the Z-5-hexadecenal, which is present in the composition in a ratio of from about 1% to about 99%. In other embodiments, the Z-11-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w and the Z-5-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w. In further embodiments, the insect pheromone composition further comprises: Z-9-hexadecenal. In still further embodiments, the insect pheromone composition further comprises: Z-9-hexadecenal and Z-7-hexadecenal.

In some embodiments, the Z-11-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w, the Z-5-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w, the Z-9-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w, and the Z-7-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w.

In some embodiments, a method of attracting an adult male Helicoverpa sp. to a locus, comprises: presenting an effective amount of the insect pheromone composition of described herein to a locus. In some embodiments, a method of attracting and killing an adult male Helicoverpa sp., comprises: presenting an effective amount of the insect pheromone composition described herein to a locus, wherein said locus also comprises a mechanism to kill the Helicoverpa sp. In some embodiments, a method of suppressing a population of Helicoverpa sp. in a given area, comprises: applying an effective amount of the insect pheromone composition disclosed herein to a locus within said area. In some embodiments, a method of suppressing a population of Helicoverpa sp. in a given area, comprises: permeating the atmosphere within said area with an effective amount of the insect pheromone composition of disclosed herein. In some embodiments, the effective amount of the insect pheromone composition is sufficient to at least partially disrupt mating within the Helicoverpa sp. population.

In some embodiments, an insect pheromone composition for modifying the behavior of a target insect, comprises: (a) a first synthetically derived insect pheromone having a chemical structure corresponding to the chemical structure of a naturally occurring insect pheromone produced by the target insect, said structure comprising formula (1),

wherein R is located on a terminal carbon of an m-end of a carbon-carbon double bond in an unsaturated hydrocarbon substrate; and (b) positional isomer of said first synthetically derived insect pheromone, said positional isomer having a chemical structure of formula (2),

wherein the positional isomer has an R located on a terminal carbon of an n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; wherein m and n are independently integers from 0 to 15, wherein a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, or e is 1, wherein b and d are independently integers from 0 to 10, the m-end and the n-end are located on opposing sides of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; and each R is independently —OH, ═O, or —OAc.

In some embodiments, the insect pheromone composition further comprises an analog of said first synthetically derived insect pheromone, said analog having a chemical structure of formula (3)

wherein the analog has an R′ located on the n-end and the m-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; wherein m and n are independently integers from 0 to 15, wherein a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, or e is 1, wherein b and d are independently integers from 0 to 10, the m-end and the n-end are located on opposing sides of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; and each R′ is independently H, —OH, ═O, —OAc, or —OOH.

In some embodiments, the insect pheromone composition further comprises:

a positional isomer of said first synthetically derived insect pheromone, said positional isomer having a chemical structure of formula (4),

wherein the positional isomer has an R located on a subterminal carbon on the m-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate thereby forming an i-end, wherein the i-end comprises a terminal carbon of the unsaturated hydrocarbon substrate; or a positional isomer of said first synthetically derived insect pheromone, said positional isomer having a chemical structure of formula 5,

wherein the positional isomer has an R located on a subterminal carbon on the n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate, thereby forming an i-end, wherein the i-end comprises a terminal carbon of the unsaturated hydrocarbon substrate; wherein m, n, and i are independently integers from 0 to 15, wherein a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, or e is 1, wherein b and d are independently integers from 0 to 10, the m-end and the n-end are located on opposing sides of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; and R is —OH, ═O, or —OAc.

In some embodiments, the sum of a, b, c, d, e, m, and n is an integer from 6 to 20. In some embodiments, the sum of a, b, c, d, e, i, m, and n is an integer from 6 to 20.

In some embodiments, the first synthetically derived pheromone has the following chemical structure:

a)

The insect pheromone composition of claim 32, wherein the first synthetically derived insect pheromone has the following chemical structure:

a)

and, wherein the positional isomer has the following chemical structure:

b)

In some embodiments, the insect pheromone composition further comprises a second synthetically derived insect pheromone having a chemical structure corresponding to the chemical structure of a naturally occurring insect pheromone produced by the target insect, wherein said second synthetically derived insect pheromone has the following chemical structure:

c)

and d) optionally, a positional isomer of said second synthetically derived insect pheromone, wherein said positional isomer is not naturally produced by the target insect.

In some embodiments, the positional isomer is present and has the following chemical structure:

The insect pheromone composition of claim 40, wherein the first synthetically derived insect pheromone has the following chemical structure:

a)

and, the positional isomer of the first synthetically derived insect pheromone has the following chemical structure:

b)

and wherein the second synthetically derived insect pheromone has the following chemical structure

c)

and

the positional isomer of the second synthetically derived insect pheromone is present and has the following chemical structure:

d)

In some embodiments, the positional isomer is not produced by the target insect. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the order Lepidoptera. In some embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the family Noctuidae or Plutellidae. In some such embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the genus Helicoverpa, Plutella, Spodoptera, or Chrysodeixis. In further embodiments, the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by an insect selected from the group consisting of Helicoverpa zea, Helicoverpa armigera, Plutella xylostella, Spodoptera frugiperda, and Chrysodeixis includens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the bioconversion of an unsaturated hydrocarbon substrate to an insect pheromone and a positional isomer.

FIG. 2 shows the bioconversion reaction of (Z)-5-hexadecene using induced strain SPV048. (Z)-5-hexedecen-1-ol elutes at 11.5 min while (Z)-11-hexadecen-1-ol elutes at 11.6 min.

FIG. 3 shows a reaction scheme for hydroxylation of asymmetric alkenes using the methods of the disclosure.

FIG. 4 shows Z-9-tetradecenyl acetate, the sex pheromone of Spodoptera frugiperda (Fall armyworm) and the positional isomer Z-5-tetradecenyl acetate.

DETAILED DESCRIPTION OF THE DISCLOSURE I. Definitions

The following definitions and abbreviations are to be used for the interpretation of the disclosure.

As used herein, the term “a” as used herein to refer to noun can refer to the singular or the plural version. Thus, a reference to a pheromone can refer to one pheromone or a more than one pheromones.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having, “contains,” “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or.”

“About” in reference to a numerical value refers to the range of values somewhat less or greater than the stated value, as understood by one of skill in the art. For example, the term “about” could mean a value ranging from plus or minus a percentage (e.g., ±1%, ±2%, ±5%, or ±10%) of the stated value. Furthermore, since all numbers, values, and expressions referring to quantities used herein are subject to the various uncertainties of measurement encountered in the art, then unless otherwise indicated, all presented values may be understood as modified by the term “about.”

The terms “engineered enzyme” and “enzyme variant” include any enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different enzymes.

The terms “engineered heme enzyme” and “heme enzyme variant” include any heme-containing enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different heme-containing enzymes.

The terms “engineered cytochrome P450” and “cytochrome P450 variant” include any cytochrome P450 enzyme comprising at least one amino acid mutation with respect to wild-type and also include any chimeric protein comprising recombined sequences or blocks of amino acids from two, three, or more different cytochrome P450 enzymes.

The term “whole cell catalyst” includes microbial cells expressing hydroxylase enzymes, wherein the whole cell catalyst displays hydroxylation activity.

As used herein, the term “metathesis reaction” refers to a catalytic reaction which involves the interchange of alkylidene units (i.e., R₂C=units) among compounds containing one or more carbon-carbon double bonds (e.g., olefinic compounds) via the formation and cleavage of the carbon-carbon double bonds. Metathesis can occur between two like molecules (often referred to as self-metathesis) and/or between two different molecules (often referred to as cross-metathesis). The product of a “metathesis reaction” can be referred to herein as a “metathesis product,” “olefinic substrate,” “unsaturated hydrocarbon” and derviation and variations thereof.

As used herein, the term “metathesis catalyst” refers to any catalyst or catalyst system that catalyzes a metathesis reaction. One of skill in the art will appreciate that a metathesis catalyst can participate in a metathesis reaction so as to increase the rate of the reaction, but is itself not consumed in the reaction.

As used herein, the term “metathesis product” refers to an olefin containing at least one double bond, the bond being formed via a metathesis reaction.

As used herein, the terms “microbial,” “microbial organism,” and “microorganism” include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a chemical.

As used herein, the term “non-naturally occurring,” when used in reference to a microbial organism or enzyme activity of the disclosure, is intended to mean that the microbial organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microbial organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous, or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary non-naturally occurring microbial organism or enzyme activity includes the hydroxylation activity described above.

As used herein, the term “natural pheromone” is intended to mean the volatile chemical or particular volatile chemical blend having a chemical structure corresponding to the chemical structure of a pheromone that is released by a particular insect for the function of chemical communication within the species. As used herein, the term “non-natural” or “non-naturally occurring,” when used in reference to a synthetic pheromone, is intended to mean a volatile chemical that is not produced by the particular insect species whose behavior is modified using said volatile chemical.

As used herein, the term “synthetically derived” when used in reference to a chemical compound is intended to indicate that the referenced chemical compound is transformed from starting material to product by human intervention. In some embodiments, a synthetically derived chemical compound can have a chemical structure corresponding an insect pheromone which is produced an insect species.

As used herein, the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The term as it is used in reference to expression of an encoding nucleic acid refers to the introduction of the encoding nucleic acid in an expressible form into the microbial organism. When used in reference to a biosynthetic activity, the term refers to an activity that is introduced into the host reference organism.

The term “heterologous” as used herein with reference to molecules, and in particular enzymes and polynucleotides. indicates molecules that are expressed in an organism other than the organism from which they originated or are found in nature, independently of the level of expression that can be lower, equal or higher than the level of expression of the molecule in the native microorganism.

On the other hand, the terms “native” and/or “endogenous” as used herein with reference to molecules, and in particular enzymes and polynucleotides, indicate molecules that are expressed in the organism in which they originated or are found in nature, independently of the level of expression that can be lower equal or higher than the level of expression of the molecule in the native microorganism. It is to be understood that expression of native enzymes or polynucleotides may be modified in recombinant microorganisms.

The term “homolog,” as used herein with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural, or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Homologs most often have functional, structural, or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homologs can be confirmed using functional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. Thus, the term “homologous proteins” is intended to mean that the two proteins have similar amino acid sequences. In certain instances, the homology between two proteins is indicative of its shared ancestry, related by evolution.

The terms “analog” and “analogous,” when used in reference to a nucleic acid or protein, include nucleic acid or protein sequences or protein structures that are related to one another in function only and are not from common descent or do not share a common ancestral sequence. Analogs may differ in sequence but may share a similar structure, due to convergent evolution. For example, two enzymes are analogs or analogous if the enzymes catalyze the same reaction of conversion of a substrate to a product, are unrelated in sequence, and irrespective of whether the two enzymes are related in structure.

As used herein, the term “alkane” refers to a straight or branched, saturated, aliphatic hydrocarbon having the number of carbon atoms indicated. The term “alkyl” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. Alkyl can include any number of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can refer to alkyl groups having up to 20 carbons atoms, such as, but not limited to heptyl, octyl, nonyl, decyl, etc. Alkanes and alkyl groups can be optionally substituted with one or more moieties selected from halo, alkenyl, and alkynyl.

As used herein, the term “alkene” and “olefin” refers to a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. The term “olefinic” refers to a composition derived from or including a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. A “terminal” alkene refers to an alkene wherein the double bond is between two carbon atoms at the end of the hydrocarbon chain (e.g., hex-1-ene). An “internal” alkene refers to an alkene wherein the double bond is between two carbon atoms that are not at the end of the hydrocarbon chain (e.g., (E)-hex-3-ene and (Z)-hex-3-ene). An “α,ω-alkenol” refers to a hydroxy-substituted terminal alkene having the formula (CH₂═CH)(CH₂)_(m)OH, wherein m is an integer ranging from 1-30, such as 2-18. The term “alkenyl” refers to a straight chain or branched hydrocarbon radical having at least 2 carbon atoms and at least one double bond. Alkenyl can include any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Alkenyl groups can have any suitable number of double bonds, including, but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groups include, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenes and alkenyl groups can be optionally substituted with one or more moieties selected from halo, alkyl, and alkynyl.

As used herein, the term “selective” refers to preferential reaction of one site on a chemical compound over another site on the compound. As a non-limiting example, selectively hydroxylating hept-3-ene (an asymmetric alkene) refers to preferentially hydroxylating one end of the hept-3-ene to form more hept-3-en-1-ol than hept-4-en-1-ol (or forming exclusively hept-3-en-1-ol without forming hept-4-en-1-ol). Selectively hydroxylating the other end of hept-3-ene would result in the formation of more hept-4-en-1-ol than hept-3-en-1-ol (or the exclusive formation of hept-4-en-1-ol without formation of hept-3-en-1-ol).

As used herein, the term “alkyne” refers to either a straight chain or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. A “terminal” alkyne refers to an alkyne wherein the triple bond is between two carbon atoms at the end of the hydrocarbon chain (e.g., hex-1-yne). An “internal” alkyne refers to an alkyne wherein the triple bond is between two carbon atoms that are not at the end of the hydrocarbon chain (e.g., hex-3-yne). The term “alkynyl” refers to either a straight chain or branched hydrocarbon radical having at least 2 carbon atoms and at least one triple bond. Alkynyl can include any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynes and alkynyl groups can be optionally substituted with one or more moieties selected from halo, alkyl, and alkenyl.

As used herein, the term “isomer” refers to a molecule having the same chemical formula as another molecule, but with a different chemical structure. That is, isomers contain the same number of atoms of each element, but have different arrangements of their atoms. Isomers include “structural isomers” and “stereoisomers.” In “structural isomers” (also referred to as “constitutional isomers”), the atoms have a different bond-sequence. Structural isomers have different IUPAC names and may or may not belong to the same functional group. This type of isomer includes skeletal isomers wherein hydrocarbon chains have variable amounts of branching, and positional isomers, which deals with the position of a functional group on a chain; and functional group isomerism, in which the molecular formula is the same but the functional group is different.

As used herein, the term “positional isomer” refers to a first compound which has the same carbon skeleton and functional group as a second compound, but differs in the location of the functional group on or in the carbon skeleton. In a particular embodiment, a positional isomer can have a functional group (e.g., hydroxyl, aldehyde, and acetyl, etc.) located on the opposite terminus of a carbon skeleton compared to a naturally occurring compound. Thus, as used herein, a positional isomer of Z-hexadec-11-en-1-al is a Z-hexadec-5-en-1-al, because the Z-hexadec-11-en-1-al and Z-hexadec-5-en-1-al are produced via hydroxylation/oxidation of opposite termini on the Z-11-hexadecene carbon skeleton as shown in FIG. 3.

In stereoisomers, the bond structure is the same, but the geometrical positioning of atoms and functional groups in space differs. This class of isomers includes enantiomers, which are isomers that are non-superimposable mirror-images of each other, and diastereomers, which are stereoisomers that are not mirror-images. Geometric isomers or cis/trans isomers are diastereomers that with a different stereochemical orientation at a bond. E/Z isomer, which are a subset of geometric isomers, are isomers with a different geometric arrangement at a double bond. Another type of isomer, conformational isomers (conformers), may be rotamers, diastereomers, or enantiomers depending on the exact compound.

The term “analog,” as used herein in reference to a chemical structure is intended to refer to compounds having a similar structure, but having a different molecular formula, e.g., a different or additional atom and/or functional group. By way of example, and not limitation, an analog of an insect pheromone can refer to molecule with two hydroxyl groups (as opposed to a single hydroxyl group required for a natural pheromone or precursor thereof) and/or an over-oxidized molecules with a carboxylic acid functional group (as opposed to an aldehyde functional group required for a natural pheromone).

An “effective amount” means that amount of the disclosed pheromone composition that is sufficient to affect desired results. An effective amount can be administered in one or more administrations. For example, an effective amount of the composition may refer to an amount of the pheromone composition that is sufficient to attract a given insect to a given locus. Further, an effective amount of the composition may refer to an amount of the pheromone composition that is sufficient to disrupt mating of a particular insect population of interest in a given locality.

II. Introduction

The present disclosure addresses a need for a safe alternative to conventional insecticides. The present disclosure provides compositions and methods for modifying the behavior of an insect using a composition comprising a pheromone.

In some aspects, the composition comprises a pheromone chemically corresponding to the pheromone naturally produced by a given insect. In some aspects, the composition comprises a pheromone chemically corresponding to the pheromone naturally produced by a given insect, along with at least one isomer of said pheromone. In various aspects, the isomer of the naturally produced insect pheromone may be a positional isomer.

In some embodiments, the insect is a pest. As used herein, the term “pest” can refer to insects that cause damage to plants, other organisms or otherwise causes a nuisance. In some embodiments, an insect pest can be attracted to a pheromone composition, e.g., by flying toward the pheromone composition or interacting with an article treated with the pheromone composition.

In various aspects, the insect that is “attracted” to the compositions taught herein may, or may not, physically contact a locus containing said pheromone composition. That is, in some aspects, the compositions taught herein are able to attract a given insect within a close proximity to a locus containing the disclosed pheromone compositions, but do not entice said insect to physically contact the locus. However, in other aspects, the compositions taught herein do entice and/or attract an insect to physically come into contact with a locus containing said pheromone compositions. In this way, inter alia, the pheromone compositions taught herein are highly “tunable” and are able to modulate the behavior (e.g., degree of attracting an insect) of an insect to a high degree, which is not associated with pheromone compositions of the prior art. Thus, the pheromone compositions taught herein, which may contain a natural insect pheromone and at least one positional isomer of said pheromone, do not merely provoke a binary “attract or not attract” response in a given insect. Rather, the pheromone compositions of the present disclosure are able to modulate the degree to which an insect is attracted along a continuous scale, depending upon, among other things, the ratio of natural pheromone to its positional isomer.

Embodiments of the present disclosure are based on the inventors' discovery of a novel methodology for the synthesis of a pheromone. The novel method includes (1) metathesis of alpha olefins to form alkenes with an internal C═C bond, (2) biohydroxylation of the product alkene via an enzymatic reaction to generate an alkenol, and (3) modification of the alkenol to an aldehyde by oxidation (MBO) or to an acetate by esterification (MBE). This method is referred to herein as MBO and MBE. The inventors' discovered synthesis, performed according to the methodology disclosed herein, yields an isomeric mixture which includes the natural pheromone and at least one isomer, e.g., a positional isomer. An unexpected and surprising result of the application of pheromone compositions comprising a synthetically derived, natural pheromone and a non-natural positional isomer was that the presence of the non-natural positional isomer in the pheromone composition modulated the behavior of the target insect. That is, while long-range attraction was maintained (i.e., upwind orienting flight), close-range attraction was eliminated. This indicates a novel partial mimic/partial antagonist response to the non-natural position isomer. Thus, the disclosure provides for pheromone compositions that comprise a natural insect pheromone, which a given target insect has evolved to recognize, along with a positional isomer of said pheromone. The combination of a natural pheromone, which would be produced by a target male insect's female counterpart—along with a positional isomer of said pheromoneμleads to a composition with markedly different behavioral modification properties, as compared to the naturally produced pheromone composition of a female insect.

Thus, in some embodiments, the pheromone compositions taught herein, can elicit a markedly different response from a natural pheromone blend, as they possess functional attributes not found in natural pheromone compositions produced by female insects of a given target species.

Furthermore, the pheromone compositions taught herein, are structurally different than any naturally occurring pheromone composition produced by a female insect of a given target species, as the pheromone compositions taught herein provide for a combination of a natural pheromone along with its positional isomer. This combination of an insect pheromone and a positional isomer of said pheromone does not occur in nature.

Pheromone

As described above, one aspect of the disclosure is a pheromone composition which can modify the behavior of an insect. A pheromone is a secreted or excreted chemical factor that triggers a social response in members of the same species. Thus, pheromones are chemicals capable of acting outside the body of the secreting individual to impact the behavior of the receiving individual. There are, inter alia, alarm pheromones, food trail pheromones, sex pheromones, aggregation pheromones, epideictic pheromones, releaser pheromones, primer pheromones, and territorial pheromones, that affect behavior or physiology.

As used herein, a pheromone can be a chemical, or a set of chemicals, that attract at least one species of insect. In some embodiments, the pheromone is a sex pheromone which attracts one sex of at least one insect. A pheromone synthesized as disclosed herein can be chemically identical to the natural substance for the target insect or it can be an isomer (e.g., a positional isomer, a constitutional isomer, or a stereoisomer, e.g, conformational isomer, geometric isomer, diastereomer, or enantiomer, etc.) or an analog of the natural pheromone. As used herein, the term “positional isomer” refers to a first compound which has the same carbon skeleton and functional group as a second compound, but differs in the location of the functional group on or in the carbon skeleton. For example, a positional isomer can have an aldehyde functional group located on the opposite terminus of the carbon skeleton compared to a naturally occurring compound. Thus, as used herein, a positional isomer of Z-hexadec-11-en-1-al is a Z-hexadec-5-en-1-al, because the Z-hexadec-5-en-1-al and Z-hexadec-5-en-1-al are produced via hydroxylation/oxidation of the opposite terminus of the Z-5-hexadecene carbon skeleton as shown in FIG. 3.

Pheromones described herein can be referred to using IUPAC nomenclature or various abbreviations and derivations. For example, (Z)-hexadec-11-en-1-al, can also be written as Z-11-hexadecen-1-al, Z-11-hexadecenal, or Z-x-y:Ald, wherein x represents the position of the double bond, and y represents the number of carbons in the hydrocarbon skeleton. Abbreviations used herein and known to those skilled art to identify functional groups on the hydrocarbon skeleton include “Ald,” indicating an aldehyde, “OH,” indicating an alcohol, and “Ac,” indicating an acetyl. Also, the number of carbons in the chain can be indicated using numerals rather than using the written name. Thus, as used herein, an unsaturated carbon chain comprised of sixteen carbons can be written as hexadecene or 16.

Non-limiting examples of C6-C20 linear insect pheromones that can be synthesized using the methodology disclosed herein are included in Table 1 below. Accordingly, a pheromone composition as described herein can include at least one of the pheromones listed in Table 1. Further, in some embodiments, the compositions taught herein comprise at least one of the pheromones listed in Table 1, along with at least one isomer thereof. In a particular embodiment, the compositions taught herein comprise at least one of the pheromones listed in Table 1, along with a positional isomer of at least one of the pheromones as listed in Table 1. In still further aspects of the disclosure, a composition may comprise only a positional isomer of a pheromone as listed in Table 1.

TABLE 1 C6-C20 Linear Pheromones Name Name (E)-2-Decen-1-ol (E,E)-10,12-Tetradecadien-1-ol (E)-2-Decenyl acetate (E,E)-10,12-Tetradecadienyl acetate (E)-2-Decenal (E,E)-10,12-Tetradecadienal (Z)-2-Decen-1-ol (E,Z)-10,12-Tetradecadienyl acetate (Z)-2-Decenyl acetate (Z,E)-10,12-Tetradecadienyl acetate (Z)-2-Decenal (Z,Z)-10,12-Tetradecadien-1-ol (E)-3-Decen-1-ol (Z,Z)-10,12-Tetradecadienyl acetate (Z)-3-Decenyl acetate (E,Z,Z)-3,8,11-Tetradecatrienyl acetate (Z)-3-Decen-1-ol (E)-8-Pentadecen-1-ol (Z)-4-Decen-1-ol (E)-8-Pentadecenyl acetate (E)-4-Decenyl acetate (Z)-8-Pentadecen-1-ol (Z)-4-Decenyl acetate (Z)-8-Pentadecenyl acetate (Z)-4-Decenal (Z)-9-Pentadecenyl acetate (E)-5-Decen-1-ol (E)-9-Pentadecenyl acetate (E)-5-Decenyl acetate (Z)-10-Pentadecenyl acetate (Z)-5-Decen-1-ol (Z)-10-Pentadecenal (Z)-5-Decenyl acetate (E)-12-Pentadecenyl acetate (Z)-5-Decenal (Z)-12-Pentadecenyl acetate (E)-7-Decenyl acetate (Z,Z)-6,9-Pentadecadien-1-ol (Z)-7-Decenyl acetate (Z,Z)-6,9-Pentadecadienyl acetate (E)-8-Decen-1-ol (Z,Z)-6,9-Pentadecadienal (E,E)-2,4-Decadienal (E,E)-8,10-Pentadecadienyl acetate (E,Z)-2,4-Decadienal (E,Z)-8,10-Pentadecadien-1-ol (Z,Z)-2,4-Decadienal (E,Z)-8,10-Pentadecadienyl acetate (E,E)-3,5-Decadienyl acetate (Z,E)-8,10-Pentadecadienyl acetate (Z,E)-3,5-Decadienyl acetate (Z,Z)-8,10-Pentadecadienyl acetate (Z,Z)-4,7-Decadien-1-ol (E,Z)-9,11-Pentadecadienal (Z,Z)-4,7-Decadienyl acetate (Z,Z)-9,11-Pentadecadienal (E)-2-Undecenyl acetate (Z)-3-Hexadecenyl acetate (E)-2-Undecenal (E)-5-Hexadecen-1-ol (Z)-5-Undecenyl acetate (E)-5-Hexadecenyl acetate (Z)-7-Undecenyl acetate (Z)-5-Hexadecen-1-ol (Z)-8-Undecenyl acetate (Z)-5-Hexadecenyl acetate (Z)-9-Undecenyl acetate (E)-6-Hexadecenyl acetate (E)-2-Dodecenal (E)-7-Hexadecen-1-ol (Z)-3-Dodecen-1-ol (E)-7-Hexadecenyl acetate (E)-3-Dodecenyl acetate (E)-7-Hexadecenal (Z)-3-Dodecenyl acetate (Z)-7-Hexadecen-1-ol (E)-4-Dodecenyl acetate (Z)-7-Hexadecenyl acetate (E)-5-Dodecen-1-ol (Z)-7-Hexadecenal (E)-5-Dodecenyl acetate (E)-8-Hexadecenyl acetate (Z)-5-Dodecen-1-ol (E)-9-Hexadecen-1-ol (Z)-5-Dodecenyl acetate (E)-9-Hexadecenyl acetate (Z)-5-Dodecenal (E)-9-Hexadecenal (E)-6-Dodecen-1-ol (Z)-9-Hexadecen-1-ol (Z)-6-Dodecenyl acetate (Z)-9-Hexadecenyl acetate (E)-6-Dodecenal (Z)-9-Hexadecenal (E)-7-Dodecen-1-ol (E)-10-Hexadecen-1-ol (E)-7-Dodecenyl acetate (E)-10-Hexadecenal (E)-7-Dodecenal (Z)-10-Hexadecenyl acetate (Z)-7-Dodecen-1-ol (Z)-10-Hexadecenal (Z)-7-Dodecenyl acetate (E)-11-Hexadecen-1-ol (Z)-7-Dodecenal (E)-11-Hexadecenyl acetate (E)-8-Dodecen-1-ol (E)-11-Hexadecenal (E)-8-Dodecenyl acetate (Z)-11-Hexadecen-1-ol (E)-8-Dodecenal (Z)-11-Hexadecenyl acetate (Z)-8-Dodecen-1-ol (Z)-11-Hexadecenal (Z)-8-Dodecenyl acetate (Z)-12-Hexadecenyl acetate (E)-9-Dodecen-1-ol (Z)-12-Hexadecenal (E)-9-Dodecenyl acetate (E)-14-Hexadecenal (E)-9-Dodecenal (Z)-14-Hexadecenyl acetate (Z)-9-Dodecen-1-ol (E,E)-1,3-Hexadecadien-1-ol (Z)-9-Dodecenyl acetate (E,Z)-4,6-Hexadecadien-1-ol (Z)-9-Dodecenal (E,Z)-4,6-Hexadecadienyl acetate (E)-10-Dodecen-1-ol (E,Z)-4,6-Hexadecadienal (E)-10-Dodecenyl acetate (E,Z)-6,11-Hexadecadienyl acetate (E)-10-Dodecenal (E,Z)-6,11-Hexadecadienal (Z)-10-Dodecen-1-ol (Z,Z)-7,10-Hexadecadien-1-ol (Z)-10-Dodecenyl acetate (Z,Z)-7,10-Hexadecadienyl acetate (E,Z)-3,5-Dodecadienyl acetate (Z,E)-7,11-Hexadecadien-1-ol (Z,E)-3,5-Dodecadienyl acetate (Z,E)-7,11-Hexadecadienyl acetate (Z,Z)-3,6-Dodecadien-1-ol (Z,E)-7,11-Hexadecadienal (E,E)-4,10-Dodecadienyl acetate (Z,Z)-7,11-Hexadecadien-1-ol (E,E)-5,7-Dodecadien-1-ol (Z,Z)-7,11-Hexadecadienyl acetate (E,E)-5,7-Dodecadienyl acetate (Z,Z)-7,11-Hexadecadienal (E,Z)-5,7-Dodecadien-1-ol (Z,Z)-8,10-Hexadecadienyl acetate (E,Z)-5,7-Dodecadienyl acetate (E,Z)-8,11-Hexadecadienal (E,Z)-5,7-Dodecadienal (E,E)-9,11-Hexadecadienal (Z,E)-5,7-Dodecadien-1-ol (E,Z)-9,11-Hexadecadienyl acetate (Z,E)-5,7-Dodecadienyl acetate (E,Z)-9,11-Hexadecadienal (Z,E)-5,7-Dodecadienal (Z,E)-9,11-Hexadecadienal (Z,Z)-5,7-Dodecadienyl acetate (Z,Z)-9,11-Hexadecadienal (Z,Z)-5,7-Dodecadienal (E,E)-10,12-Hexadecadien-1-ol (E,E)-7,9-Dodecadienyl acetate (E,E)-10,12-Hexadecadienyl acetate (E,Z)-7,9-Dodecadien-1-ol (E,E)-10,12-Hexadecadienal (E,Z)-7,9-Dodecadienyl acetate (E,Z)-10,12-Hexadecadien-1-ol (E,Z)-7,9-Dodecadienal (E,Z)-10,12-Hexadecadienyl acetate (Z,E)-7,9-Dodecadien-1-ol (E,Z)-10,12-Hexadecadienal (Z,E)-7,9-Dodecadienyl acetate (Z,E)-10,12-Hexadecadienyl acetate (Z,Z)-7,9-Dodecadien-1-ol (Z,E)-10,12-Hexadecadienal (Z,Z)-7,9-Dodecadienyl acetate (Z,Z)-10,12-Hexadecadienal (E,E)-8,10-Dodecadien-1-ol (E,E)-11,13-Hexadecadien-1-ol (E,E)-8,10-Dodecadienyl acetate (E,E)-11,13-Hexadecadienyl acetate (E,E)-8,10-Dodecadienal (E,E)-11,13-Hexadecadienal (E,Z)-8,10-Dodecadien-1-ol (E,Z)-11,13-Hexadecadien-1-ol (E,Z)-8,10-Dodecadienyl acetate (E,Z)-11,13-Hexadecadienyl acetate (E,Z)-8,10-Dodecadienal (E,Z)-11,13-Hexadecadienal (Z,E)-8,10-Dodecadien-1-ol (Z,E)-11,13-Hexadecadien-1-ol (Z,E)-8,10-Dodecadienyl acetate (Z,E)-11,13-Hexadecadienyl acetate (Z,E)-8,10-Dodecadienal (Z,E)-11,13-Hexadecadienal (Z,Z)-8,10-Dodecadien-1-ol (Z,Z)-11,13-Hexadecadien-1-ol (Z,Z)-8,10-Dodecadienyl acetate (Z,Z)-11,13-Hexadecadienyl acetate (Z,E,E)-3,6,8-Dodecatrien-1-ol (Z,Z)-11,13-Hexadecadienal (Z,Z,E)-3,6,8-Dodecatrien-1-ol (E,E)-10,14-Hexadecadienal (E)-2-Tridecenyl acetate (Z,E)-11,14-Hexadecadienyl acetate (Z)-2-Tridecenyl acetate (E,E,Z)-4,6,10-Hexadecatrien-1-ol (E)-3-Tridecenyl acetate (E,E,Z)-4,6,10-Hexadecatrienyl acetate (E)-4-Tridecenyl acetate (E,Z,Z)-4,6,10-Hexadecatrien-1-ol (Z)-4-Tridecenyl acetate (E,Z,Z)-4,6,10-Hexadecatrienyl acetate (Z)-4-Tridecenal (E,E,Z)-4,6,11-Hexadecatrienyl acetate (E)-6-Tridecenyl acetate (E,E,Z)-4,6,11-Hexadecatrienal (Z)-7-Tridecenyl acetate (Z,Z,E)-7,11,13-Hexadecatrienal (E)-8-Tridecenyl acetate (E,E,E)-10,12,14-Hexadecatrienyl acetate (Z)-8-Tridecenyl acetate (E,E,E)-10,12,14-Hexadecatrienal (E)-9-Tridecenyl acetate (E,E,Z)-10,12,14-Hexadecatrienyl acetate (Z)-9-Tridecenyl acetate (E,E,Z)-10,12,14-Hexadecatrienal (Z)-10-Tridecenyl acetate (E,E,Z,Z)-4,6,11,13- Hexadecatetraenal (E)-11-Tridecenyl acetate (E)-2-Heptadecenal (Z)-11-Tridecenyl acetate (Z)-2-Heptadecenal (E,Z)-4,7-Tridecadienyl acetate (E)-8-Heptadecen-1-ol (Z,Z)-4,7-Tridecadien-1-ol (E)-8-Heptadecenyl acetate (Z,Z)-4,7-Tridecadienyl acetate (Z)-8-Heptadecen-1-ol (E,Z)-5,9-Tridecadienyl acetate (Z)-9-Heptadecenal (Z,E)-5,9-Tridecadienyl acetate (E)-10-Heptadecenyl acetate (Z,Z)-5,9-Tridecadienyl acetate (Z)-11-Heptadecen-1-ol (Z,Z)-7,11-Tridecadienyl acetate (Z)-11-Heptadecenyl acetate (E,Z,Z)-4,7,10-Tridecatrienyl (E,E)-4,8-Heptadecadienyl acetate acetate (E)-3-Tetradecen-1-ol (Z,Z)-8,10-Heptadecadien-1-ol (E)-3-Tetradecenyl acetate (Z,Z)-8,11-Heptadecadienyl acetate (Z)-3-Tetradecen-1-ol (E)-2-Octadecenyl acetate (Z)-3-Tetradecenyl acetate (E)-2-Octadecenal (E)-5-Tetradecen-1-ol (Z)-2-Octadecenyl acetate (E)-5-Tetradecenyl acetate (Z)-2-Octadecenal (E)-5-Tetradecenal (E)-9-Octadecen-1-ol (Z)-5-Tetradecen-1-ol (E)-9-Octadecenyl acetate (Z)-5-Tetradecenyl acetate (E)-9-Octadecenal (Z)-5-Tetradecenal (Z)-9-Octadecen-1-ol (E)-6-Tetradecenyl acetate (Z)-9-Octadecenyl acetate (Z)-6-Tetradecenyl acetate (Z)-9-Octadecenal (E)-7-Tetradecen-1-ol (E)-11-Octadecen-1-ol (E)-7-Tetradecenyl acetate (E)-11-Octadecenal (Z)-7-Tetradecen-1-ol (Z)-11-Octadecen-1-ol (Z)-7-Tetradecenyl acetate (Z)-11-Octadecenyl acetate (Z)-7-Tetradecenal (Z)-11-Octadecenal (E)-8-Tetradecenyl acetate (E)-13-Octadecenyl acetate (Z)-8-Tetradecen-1-ol (E)-13-Octadecenal (Z)-8-Tetradecenyl acetate (Z)-13-Octadecen-1-ol (Z)-8-Tetradecenal (Z)-13-Octadecenyl acetate (E)-9-Tetradecen-1-ol (Z)-13-Octadecenal (E)-9-Tetradecenyl acetate (E)-14-Octadecenal (Z)-9-Tetradecen-1-ol (E,Z)-2,13-Octadecadien-1-ol (Z)-9-Tetradecenyl acetate (E,Z)-2,13-Octadecadienyl acetate (Z)-9-Tetradecenal (E,Z)-2,13-Octadecadienal (E)-10-Tetradecenyl acetate (Z,E)-2,13-Octadecadienyl acetate (Z)-10-Tetradecenyl acetate (Z,Z)-2,13-Octadecadien-1-ol (E)-11-Tetradecen-1-ol (Z,Z)-2,13-Octadecadienyl acetate (E)-11-Tetradecenyl acetate (E,E)-3,13-Octadecadienyl acetate (E)-11-Tetradecenal (E,Z)-3,13-Octadecadienyl acetate (Z)-11-Tetradecen-1-ol (E,Z)-3,13-Octadecadienal (Z)-11-Tetradecenyl acetate (Z,E)-3,13-Octadecadienyl acetate (Z)-11-Tetradecenal (Z,Z)-3,13-Octadecadienyl acetate (E)-12-Tetradecenyl acetate (Z,Z)-3,13-Octadecadienal (Z)-12-Tetradecenyl acetate (E,E)-5,9-Octadecadien-1-ol (E,E)-2,4-Tetradecadienal (E,E)-5,9-Octadecadienyl acetate (E,E)-3,5-Tetradecadienyl acetate (E,E)-9,12-Octadecadien-1-ol (E,Z)-3,5-Tetradecadienyl acetate (Z,Z)-9,12-Octadecadienyl acetate (Z,E)-3,5-Tetradecadienyl acetate (Z,Z)-9,12-Octadecadienal (E,Z)-3,7-Tetradecadienyl acetate (Z,Z)-11,13-Octadecadienal (E,Z)-3,8-Tetradecadienyl acetate (E,E)-11,14-Octadecadienal (E,Z)-4,9-Tetradecadienyl acetate (Z,Z)-13,15-Octadecadienal (E,Z)-4,9-Tetradecadienal (Z,Z,Z)-3,6,9-Octadecatrienyl acetate (E,Z)-4,10-Tetradecadienyl acetate (E,E,E)-9,12,15-Octadecatrien-1-ol (E,E)-5,8-Tetradecadienal (Z,Z,Z)-9,12,15-Octadecatrienyl acetate (Z,Z)-5,8-Tetradecadien-1-ol (Z,Z,Z)-9,12,15-0ctadecatrienal (Z,Z)-5,8-Tetradecadienyl acetate (Z,Z)-5,8-Tetradecadienal (E,E)-8,10-Tetradecadien-1-ol (E,E)-8,10-Tetradecadienyl acetate (E,E)-8,10-Tetradecadienal (E,Z)-8,10-Tetradecadienyl acetate (E,Z)-8,10-Tetradecadienal (Z,E)-8,10-Tetradecadien-1-ol (Z,E)-8,10-Tetradecadienyl acetate (Z,Z)-8,10-Tetradecadienal (E,E)-9,11-Tetradecadienyl acetate (E,Z)-9,11-Tetradecadienyl acetate (Z,E)-9,11-Tetradecadien-1-ol (Z,E)-9,11-Tetradecadienyl acetate (Z,E)-9,11-Tetradecadienal (Z,Z)-9,11-Tetradecadien-1-ol (Z,Z)-9,11-Tetradecadienyl acetate (Z,Z)-9,11-Tetradecadienal (E,E)-9,12-Tetradecadienyl acetate (Z,E)-9,12-Tetradecadien-1-ol (Z,E)-9,12-Tetradecadienyl acetate (Z,E)-9,12-Tetradecadienal (Z,Z)-9,12-Tetradecadien-1-ol (Z,Z)-9,12-Tetradecadienyl acetate

In some aspects, pheromone compositions taught in this disclosure comprise at least one pheromone listed in Table 2 and a positional isomer thereof to modulate the behavior of an insect listed in Table 2. By changing the ratios of a pheromone as listed in Table 2 and a positional isomer thereof in a given composition, the disclosure provides for a highly tunable insect behavior modifying composition.

TABLE 2 Exemplary compounds that can be synthesized, combined into compositions, and used according to methods described in the present disclosure. Example of Biological Name Structure importance (Z)-3-hexanol

See, Sugimoto etal. (2014) (Z)-3-nonen-1-ol

West Indian Fruity Fly male sex pheromone (Z)-5-decen-1-ol

(Z)-5-decenyl acetate

Agrotis segetum sex pheromone component (E)-5-decen-1-ol

Anarsia lineatella sex pheromone component (E)-5-decenyl acetate

Anarsia lineatella sex pheromone component (Z)-7-dodecen-1-ol

(Z)-7-dodecenyl acetate

Pseudoplusia includens sex pheromone Agrotis segetum sex pheromone component (E)-8-dodecen-1-ol

Citrus Fruit Moth sex pheromone (E)-8-dodecenyl acetate

Grapholitha molesta, Ecdytolopha aurantiana sex pheromone component (Z)-8-dodecen-1-ol

Grapholitha molesta, Ecdytolopha aurantiana sex pheromone component (Z)-8-dodecenyl acetate

Grapholitha molesta sex pheromone component (Z)-9-dodecen-1-01

(Z)-9-dodecenyl acetate

Eupoecilia ambiguella sex pheromone (Z)-9-tetradecen-1-01

(Z)-9-tetradecenyl acetate

Pandemis pyrusana, Naranga aenescens, Agrotis segetum sex pheromone component (Z)-11-tetraceden-1-ol

(Z)-11-tetracedenyl acetate

Pandemis pyrusana, Choristoneura roseceana sex pheromone component (E)-11-tetradecen-1-ol

(E)-11-tetradecenyl acetate

Choristoneura roseceana, Crocidolomia pavonana sex pheromone component (Z)-7-hexadecen-1-ol

(Z)-7-hexadecenal

Diatraea considerata sex pheromone component (Z)-9-hexadecen-1-ol

(Z)-9-hexadecenal

Helicoverpa zea, Helicoverpa armigera, Heliothis virescens sex pheromone component (Z)-9-hexadecenyl acetate

Naranga aenescens sex pheromone component (Z)-11-hexadecen-1-ol

(Z)-11-hexadecenal

Platyptila carduidactyla, Heliothis virescens sex pheromone Helicoverpa zea, Helicoverpa armigera, Plutella xylostella, Diatraea considerate, Diatraea grandiosella, Diatraea saccharalis, Acrolepiopsis assectella sex pheromone component (Z)-11-hexadecenyl acetate

Discestra trifolii sex pheromone Heliothis virescens, Plutella xylostella, Acrolepiopsis assectella, Crocidolomia pavonana, Naranga aenescens sex pheromone component (Z)-13-octadecen-1-ol

(Z)-13-octadecenal

Diatraea considerata, Diatraea grandiosella sex pheromone component Ac = —(CO)CH₃

Pheromones have the potential to challenge conventional approaches to agricultural insect control. Since their discovery in the late 1950s, these molecules have shown efficacy in reducing insect populations through sensory disruption and a subsequent reduction in mating frequency via a non-toxic mode of action.

Insect pheromones can be used in a variety of insect control strategies, including mating disruption, attract-and-kill, and mass trapping. These strategies have proven to be effective, selective (e.g., they do not harm beneficial insects, such as bees and lady bugs), and safe (e.g., the compounds are generally biodegradable and do not accumulate in the food chain).

The selectivity of pheromones allows farmers to control the population of the target pest causing minimal disruption to the ecology in the field. Because pheromones act via non-toxic mating disruption, they can be used to manage pests that have evolved resistance to chemical or transgenic insecticides.

This organic form of insect control has enjoyed success in permanent crops worldwide, particularly in Washington State apple orchards where adoption rates are greater than 90%. However, only <20 insect pests worldwide are currently controlled using pheromone solutions (e.g., mating disruption, attract-and-kill, mass trapping), and only 0.05% of global agricultural land employs pheromones. The limited use of pheromones is an unfortunate result of the high cost of synthesizing pheromones is very high, with industrial scale active ingredient (AI) prices ranging from $500 to $15,000 per kg, which prohibits widespread use of this sustainable technology beyond high-value crops.

Described herein are pheromone compositions synthesized using a novel enzymatic biohydroxylation step, which yields a pheromone at a fraction of the cost of conventional methodology. In some embodiments, the pheromone synthesized according to this methodology is a positional isomer of the natural pheromone. In one such embodiment, the positional isomer is not naturally produced by a female insect, but a male insect of the same species surprisingly responds to a composition which includes the positional isomer. This is surprising, given the fact that male insects are highly evolved to sense and respond to pheromone produced by their potential female mate. Accordingly, in some embodiments, a pheromone composition comprising: (a) an insect pheromone having a chemical structure identical to that of a pheromone produced by an insect pest, and (b) a positional isomer of said insect pheromone, can be used to modify the behavior of an insect pest.

The inclusion of a positional isomer in a pheromone composition comprising a synthetically derived natural pheromone can have at least four possible outcomes: (1) Inert—the isomer acts as a diluent, and the behavior of the target insect is not influenced; (2) Antagonist—the isomer acts as an inhibitor and blocks the response of the target insect to the natural pheromone; (3) Mimic—the isomer provides the same biological activity as the natural pheromone; and (4) Partial Mimic/Partial Antagonist—the isomer elicits an upwind flight response from the target insect, but the insect does not contact or land on a lure coated with the positional isomer.

In one embodiment, the target insect is member of the order Lepidoptera. In some embodiments, the composition is utilized to bring about mating disruption in the lepidopteran population, which subsequently leads to a decline in the population.

Lepidoptera

Lepidoptera is the second largest order in the class Insecta. The order Lepidoptera include the following families of butterflies: Nymphalidae, Danaidae, Pieridae, Papilionidae, Lycaenidae, Hesperiidae (e.g., Epargyreus clarus). The order also includes the following families of moths: Tineidae (e.g., (Tineola bisselliella, and Tinea pellionella), Gelechiidae (e.g., Sitotroga cerealella and Pectinophora gossypiella), Sesiidae (e.g., Synanthedon exitiosa and Melittia cucurbitae), Tortricidae (e.g., Cydia pomonella and Grapholita molesta). Pyralidae (e.g., Ostrinia nubilalis, Plodia interpunctella, and Galleria mellonella), Geometridae (e.g., Operophtera brumata and Alsophila pometaria), Lasiocampidae (e.g., Malacosoma Americana and Malacosoma disstria). Satumiidae (e.g., Hyalophora cecropiaa and Actias luna), Sphingidae (e.g., Manduca sexta and Manduca quinquemaculata), Arctiidae (e.g., Hyphantria cunea), Lymantriidae (e.g., Lymantria dispar and Euproctis chrysorrhoea), Noctuidae (e.g., Spodoptera frugiperda, Agrotis Ipsilon, Trichoplusia ni, Chrysodeixis includes, Helicoverpa zea, and Helicoverpa armigera), and Plutellidae (e.g., Plutella xylostella).

The larvae of many lepidopteran species, which are commonly referred to as caterpillars, are major pests in agriculture. In many lepidopteran species, the female may produce anywhere from 200 to 600 eggs; and some species produce up to 30,000 eggs in one day. Unmitigated, the larvae can affect acres of vegetation. In fact, the larvae of Lepidoptera are probably more destructive to agricultural crops and forest trees than any other group of insects.

Some of the major pests of the order Lepidoptera include members of the families Noctuidae and Plutellidae. The larvae of the Noctuidae genus Spodoptera (including armyworm), Helicoverpa (including corn earworm and cotton bollworm), Chrysodeixis (including soybean looper) and larvae of the Plutellidae genus Plutella (including diamondback moth) can cause extensive damage to valuable crops.

Helicoverpa zea is known as the corn earworm; the polyphagous larva are known to cause damage to a variety of crops, including: corn, tomato, artichoke, asparagus, cabbage, cantaloupe, collards, cowpea, cucumber, eggplant, lettuce, lime bean, melon, okra, pea, pepper, potato, pumpkin, snap bean, spinach, squash, sweet potato, watermelon, soybean, as non-limiting examples.

Helicoverpa armigera is commonly referred to as the cotton bollworm; the polyphagous larva are known to cause damage to a variety of crops, including: tomato, cotton, pigeon pea, chickpea, sorghum, cowpea, groundnut, okra, peas, field beans, soybeans, lucerne, a variety of legumes, tobacco, potatoes, maize, flax, Dianthus, Rosa, Pelargonium, Chrysanthemum, Lavandula angustifolia, fruit trees, forest trees, and a range of vegetable crops, as non-limiting examples.

Plutella xylostella is known as the diamondback moth and is a worldwide pest; it is known to feed on cruciferous vegetables, including: broccoli, Brussels sprouts, cabbage, Chinese cabbage, cauliflower, collard, kale, kohlrabi, mustard, radish, turnip, and watercress.

Spodoptera frugiperda, known as the fall armyworm, has been reported to damage field crops, including: alfalfa, barley, Bermuda grass, buckwheat, cotton, clover, corn, oat, millet, peanut, rice, ryegrass, sorghum, sugarbeet, sudangrass, soybean, sugarcane, timothy, tobacco, and wheat, sweet corn, apple, grape, orange, papaya, peach, strawberry and a number of flowers.

Chrysodeixis includens is a type of moth whose larva is known to damage crops, including: soybeans, goldenrod, lettuce, sweet potato, peanut, cotton, tomato, brassicas (cabbage, kale, broccoli), pea, tobacco, and cocklebur.

Today, lepidopteran pests are predominantly controlled by pyrethroid, organophosphate, and carbamate insecticide sprays. Organophosphates and carbamates have demonstrated carcinogenic and neurotoxic effects in humans, while pyrethroids and organophosphates may unintentionally harm beneficial insects or sensitive vertebrates like amphibians, and fish. Conversely, lepidopteran pheromones present no known risks to humans or the environment. These non-toxic compounds may serve as a substitute for conventional pesticides, reducing the amount of chemical exposure to consumers, farm laborers, and the environment.

Insects of the order Lepidoptera produce pheromones which generally consist of unbranched, oxyfunctionalized long-chain olefins containing one to three double bonds. Lepidopteran pheromones, which are naturally occurring compounds, or identical or substantially similar synthetic compounds, are designated by an unbranched aliphatic chain (between 9 and 18 carbons) ending in an alcohol, aldehyde, or acetate functional group and containing up to 3 double bonds in the aliphatic backbone. For examples, the sex pheromones of Helicoverpa zea, Helicoverpa armigera, Plutella xylostella, and Chrysodeixis includes insects typically include one or more aliphatic aldehyde compounds having from 10 to 16 carbon atoms (e.g., 7-hexadecenal, 11-hexadecenal, 13-octadecenal, and the like). Other insects, such as Spodoptera frugiperda, recognize pheromones that are aliphatic acetate compounds having from 10 to 16 carbon atoms (e.g., decyl acetate, decenyl acetate, decadienyl acetate, undecyl acetate, undecenyl acetate, dodecyl acetate, dodecenyl acetate, dodecadienyl acetate, tridecyl acetate, tridecenyl acetate, tridecadienyl acetate, tetradecyl acetate, tetradecenyl acetate, tetradecadienyl acetate, and the like).

Variation in the location, cis/trans selectivity, level of unsaturation along the chain, and chain length results in a diverse set of pheromones that facilitate species specific communication. These pheromones are used to attract a mate, sometimes at long distances.

The generally accepted natural pheromones produced by female Helicoverpa zea, Helicoverpa armigera, Plutella xylostella and Chrysodeixis includens are shown in the table below. Thus, aspects of the disclosure provide for pheromone compositions comprised of synthetically derived natural pheromone blends according to the table below along with various ratios of positional isomers. However, it should be noted that there can be other minor components produced by these insects as well. The below table merely lists the pheromone compositions produced by these insects, as they are commonly understood in the scientific literature. See, e.g., http://www.pherobase.com/database/species/species-Helicoverpa-zea.php; Halfhill, J. E. and McDonough, L. M. Southwest Entomol., 1985. 10; 176-180; Pope M M, Gaston L K, and Baker T C. (1984) Composition, quantification, and periodicity of sex pheromone volatiles from individual Heliothis zea females. J Insect Physiol 30:943-945; http://www.pherobase.com/database/species/species-Helicoverpa-armigera.php; Zhang, J. P., et al. J. Insect Physiol. (2012) 58:1209-1216; http://www.pherobase.com/database/species/species-Plutella-xylostella.php; Lin, Y. M., et al., Bull. Inst. Sool. Acad. Sin. 21:121-127; Chisholm M D, Underhill E W, and Steck W F. (1979) Field trapping of the diamondback moth Plutella xylostella using synthetic sex attractants. Environmental Entomology 8:516-518; http://www.pherobase.com/database/species/species-Spodoptera-frugiperda.php; Meagher, R. L. and Mitchell, E. R. Fla. Entomol., 1998. 81:556-559; Tumlinson J H, Mitchell E R, Teal P E A, Heath R R, and Mengelkoch L J (1986) Sex pheromone of fall armyworm, Spodoptera frugiperda (J. E. Smith). J Chem Ecol 12:1909-1926; http://www.pherobase.com/database/species/species-Pseudoplusia-includens.php; Tumlinson, J. H, et al., Environ. Entomol., 1972. 1:466-468; Linn C E, Du J, Hammond A, and Roelofs W L. (1987) Identification of unique pheromone components for soybean looper moth Pseudoplusia includens. J Chem Ecol 13:1351-1360; Cork A, Beevor P S, Hall D R, Nesbitt B F, Arida G S, and Mochida O. (1985). Components of the female sex pheromone of the yellow stem borer, Scirpophaga incertulas. Entomol. Exp. Appl. 37:149-153. Jiao X-G, Xuan W-J, Sheng C-F (2005) Mass trapping of the overwintering generation stripped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae) with the synthetic sex pheromone in northeastern China. Acta Entomologica Sinica 48:370-374; McLaughlin J and R Heath (1989) Field trapping and observations of male Velvetbean caterpillar moths and trapping of Mocis spp. (Lepidoptera: Noctuidae: Catacolinae) with calibrated formulations of sex pheromone. Environmental Entomology 18:933-938

Natural Pest Pest Pheromone (Latin Name(s)) (English Name(s)) Blend %^(†) Helicoverpa zea Corn earworm Z11-hexadecenal 98 Z9-hexadecenal¹ 2 Helicoverpa armigera Cotton bollworm Z11-hexadecenal 98.3 Corn earworm Z9-hexadecenal 1.4 Old world bollworm Z9-tetradecenal² 0.3 Plutella xylostella Diamondback moth Z11-hexadecenal 70 Z11-hexadecenyl 30 acetate³ Spodoptera frugiperda Fall armyworm Z9-tetradecenyl 96.6 acetate Z7-dodecenyl 3.4 acetate⁴ Chrysodeixis includens Soybean looper Z7-dodecenyl 100 (Pseudoplusia acetate⁵ includens) Scirpophaga Yellow stem borer Z11-hexadecenal 75 incertulas Rice stem borer Z9-hexadecenal⁶ 25 Chilo suppressalis Asiatic (or Striped) Z11-hexadecenal 82 rice stem borer Z13-octadecenal 10 Z9-hexadecenal⁷ 8 Anticarsia gemmatalis Velvetbean Z3,6,9-eicosatriene 62.5 caterpillar Velvetbean moth Z3,6,9- 37.5 heneicosatriene⁸ ^(†)Ratios vary between regional populations and studies. The ratios reported here are based on either recent citations, more commonly cited blends, or historically accepted blends.

It is common for an individual pheromone to appear in multiple insects. For example, (Z)-11-hexadecenal is the main pheromone component for not only the corn earworm, but also the tobacco budworm, diamondback moth, and the rice stem borer.

As discussed above, pheromones can be used to manage pests. Accordingly, described herein are pheromone compositions and methods of use thereof to modulate the behavior of pests, e.g., by disrupting mating behavior.

In some embodiments, a pheromone described in Table 1 or Table 2 can be synthesized using the methods and synthetic schemes described herein. In aspects, positional isomers of the pheromones listed in Table 1 or Table 2 are produced by the synthetic schemes disclosed herein. Accordingly, a pheromone composition as described herein can include at least one of the pheromones listed in Table 1 or Table 2, along with at least one isomer thereof. In a particular embodiment, the compositions taught herein comprise at least one of the pheromones listed in Table 1 or Table 2, along with a positional isomer of at least one of the pheromones as listed in Table 1 or Table 2.

In exemplary embodiments, isomers of hexadecen-1-al can be synthesized for use in pheromone compositions. In the present disclosure, Z-hexadac-11-en-1-al, Z-11hexadacen-1-al, Z-11-hexadacenal, Z-hexadac-11-enal and Z-11-16:AL, are used synonymously, and similar variations can be used for other phenomes described herein. In exemplary embodiments, a Z-hexadac-11-en-1-al and a positional can be synthesized for use in pheromone compositions to modify the behavior of insect of the order Lepidoptera (e.g., Helicoverpa. zea,). In one such exemplary embodiment, the positional isomer of Z-hexadac-11-en-1-al is Z-hexadac-5-en-1-al (FIG. 1). In some embodiments, Z-hexadac-11-en-1-yl acetate can and a positional isomer can synthesized for use in pheromone compositions to modulate the behavior of an insect of the order Lepidoptera (e.g., Plutella xylostella and Spodoptera frugiperda). In one such embodiment, the positional isomer of Z-hexadac-11-en-1-yl acetate is Z-hexadac-5-en-1-yl acetate. In another embodiment, Z-hexadac-9-en-1-al and a positional isomer can be synthesized for use in pheromone compositions to modify the behavior of an insect of the order Lepidoptera (e.g., Helicoverpa armigera and Helicoverpa. zea). In one such exemplary embodiment, the positional isomer of Z-hexadac-9-en-1-al is Z-hexadac-7-en-1-al. In yet another embodiment, Z-tetradec-9-en-1-al and a positional isomer can by synthesized for use in pheromone compositions to modulate the behavior of an insect of the order Lepidoptera (e.g., Spodoptera frugiperda). In one such exemplary embodiment, the positional isomer of Z-tetradec-9-en-1-al is Z-hexadac-5-en-1-al. In still another embodiments, Z-tetradec-9-en-1-yl acetate and its positional isomer can by synthesized for use in pheromone compositions to modify the behavior of an insect of the order Lepidoptera (e.g., Spodoptera frugiperda). In one such exemplary embodiment, the positional isomer of Z-tetradec-9-en-1-yl acetate is Z-hexadac-5-en-1-yl acetate (FIG. 4). In an even further embodiment, Z-dodec-7-en-1-yl acetate and positional isomer can by synthesized for use in pheromone compositions to modify the behavior of an insect of the order Lepidoptera (e.g., Chrysodeixis includens). In one such exemplary embodiment, the positional isomer of Z-dodec-7-en-1-yl acetate is Z-dodec-5-en-1-yl acetate.

The present disclosure is based in part on the inventors' unexpected discovery that a pheromone composition including a synthetically derived natural pheromone and a positional isomer thereof can be used to modulate the response of a target insect relative to the response of the target insect elicited by a natural pheromone or natural pheromone blend.

III. Compositions General Synthetic Route to Produce Pheromone Compositions

The present disclosure describes several methods for the synthesis of terminally oxyfunctionalized alkenes. Said methods are described in detail below and are generally applicable to the synthesis of various compounds, including but not limited to those shown in Table 1.

Some embodiments of the disclosure provide methods for synthesizing olefinic alcohol products wherein the olefinic alcohol product is a pheromone. In some embodiments, the olefinic alcohol product is selected from the alcohols in Table 1. Pheromones containing aldehyde functional groups can also be prepared using the olefinic alcohol products as intermediates. In such cases, the methods of the disclosure generally include oxidizing the olefin alcohol product to form an alcohol product. In some embodiments, the olefinic aldehyde product is selected from the aldehydes in Table 1.

Pheromones containing ester functional groups can also be prepared using the olefinic alcohol products as intermediates. In such cases, the methods of the disclosure generally include esterifying the olefinic alcohol product to form an olefinic ester product. In some embodiments, the olefinic ester product is an acetate ester. In some embodiments, the olefinic ester product is selected from the esters in Table 1 or Table 2.

Useful unsaturated fatty acids and related compounds can also be prepared using the olefinic alcohol products as intermediates. In such cases, the methods of the disclosure generally include oxidizing the olefinic alcohol product to form an olefinic acid product.

The synthetic strategies disclosed herein chiefly rely on the ability of hydroxylases to terminally hydroxylate hydrocarbon substrates such as linear alkenes. Linear alkenes and other hydrocarbon substrates can be synthesized via any route, including but not limited to olefin metathesis, Wittig olefination, or alkyne substitution followed by partial hydrogenation. The hydroxylation products can further be modified via any method, including—but not limited to—oxidation, esterification, and olefin metathesis, to produce the desired end products (Scheme 1). Deviations from this general scheme are also disclosed.

MBO or MBE Synthesis

In an exemplary embodiment, the synthesis route (Scheme 2) consists of (1) metathesis of alpha olefins to form alkenes with an internal C═C bond, (2) biohydroxylation of the product alkene via an enzymatic reaction to generate an alkenol, and (3) modification of the alkenol to an aldehyde by oxidation (MBO) or to an acetate by esterification (MBE). This short and concise route can potentially capture a large segment of all lepidopteran pheromones. Further, synthesis of any insect pheromone and its positional isomer can be achieved through altering the length of the alpha olefins used in the metathesis step and finding an enzyme catalyst capable of acting on a range of alkenes. Biohydroxylation of different terminal carbons on an olefinic substrate (and subsequent oxidation/esterification if necessary) will generate a mixture of pheromones having a chemical structure of an insect sex pheromone produced by an insect and positional isomers of said sex pheromone. Thus, the disclosure is not limited to producing compositions comprising lepidopteran pheromones and positional isomers thereof; rather, the methods of the disclosure can produce any insect pheromone and any positional isomers thereof, for utilization in the disclosed compositions.

Synthesis of Terminal Alkenols Via Metathesis and Hydroxylation

In one aspect, the disclosure provides a method for synthesizing an olefinic alcohol product that includes incubating an unsaturated hydrocarbon substrate with an enzyme capable of independently hydroxylating a first terminal carbon of a first unsaturated hydrocarbon substrate and a second terminal carbon of a second unsaturated hydrocarbon substrate to form a mixture of an unsaturated hydrocarbon alcohol. The hydrocarbon alcohol can be further converted via oxidation, acetylation, or esterification as disclosed herein or according to methods known to those skilled in the art.

FIG. 1 shows a first unsaturated hydrocarbon 101 having a first end 103 and a second end 104, and a second unsaturated hydrocarbon 102 having a first end 105 and a second end 106. Biohydroxylation is performed by an enzyme catalyst 107, which independently oxidizes the second end 106 of the second substrate 102 to produce a naturally occurring sex pheromone 109 (or precursor thereof), and the first end 103 of the first substrate 101 to produce a positional isomer 108 of the sex pheromone (or precursor thereof).

Synthesis of Unsaturated Hydrocarbon Substrate for Subsequent Biohydroxylation to Produce Pheromone Composition Hydroxylation of Asymmetric Alkenes

In some embodiments, the method for synthesizing an oxyfunctionalized alkene includes a combination of metathesis and terminal hydroxylation as shown in Scheme 3. In this process, terminal alkenes of different lengths are combined to generate asymmetric alkenes, which are then subjected to biohydroxylation conditions to afford the desired alkenol products.

Methods including hydroxylation of asymmetric alkenes can be conducted with alkenes of any suitable length. In some embodiments, the asymmetric olefinic alcohol product is a C4-C30 olefinic alcohol product. In such embodiments, the sum of the subscripts m and n shown in Scheme 3 will bring the total number of carbon atoms in a particular asymmetric olefinic alcohol product to 4-30, when added to the number of the non-subscripted carbon atoms shown in the structure for the asymmetric olefinic alcohol product. In such embodiments, for example, subscript m in Scheme 5 can be an integer from 8-18 and subscript n in Scheme 3 can be a different integer from 0-8, bringing the total number of the carbons in the asymmetric olefinic substrate to 4-30. When m is 9 and n is 3, the route depicted in Scheme 3 provides (E/Z)-hexadec-11-en-1-ol as the target product. In some embodiments, the asymmetric olefinic alcohol product is a C4-C20 olefinic alcohol product. The asymmetric olefinic alcohol can contain, for example, 4-20 carbon atoms, or 8-20 carbon atoms, or 12-20 carbon atoms, or 16-20 carbon atoms.

In some embodiments, for example, m is 0 and n is 4; or m is 1 and n is 3; or m is 3 and n is 1; or m is 4 and n is 0; or m is 0 and n is 5; or m is 1 and n is 4; or m is 2 and n is 3; or m is 3 and n is 2; or m is 4 and n is 1; or m is 5 and n is 0; or m is 0 and n is 6; or m is 1 and n is 5; or m is 2 and n is 4; or m is 4 and n is 2; or m is 5 and n is 1; or m is 6 and n is 0; or m is 0 and n is 7; or m is 1 and n is 6; or m is 2 and n is 5; or m is 3 and n is 4; or m is 4 and n is 3; or m is 5 and n is 2; or m is 6 and n is 1; or m is 7 and n is 0; or m is 0 and n is 8; or m is 1 and n is 7; or m is 2 and n is 6; or m is 3 and n is 5; or m is 5 and n is 3; or m is 6 and n is 2; or m is 7 and n is 1; or m is 8 and n is 0; or m is 0 and n is 9; or m is 1 and n is 8; or m is 2 and n is 7; or m is 3 and n is 6; or m is 4 and n is 5; or m is 5 and n is 4; or m is 6 and n is 3; or m is 7 and n is 2; or m is 8 and n is 1; or m is 9 and n is 0; or m is 0 and n is 10; or m is 1 and n is 9; or m is 2 and n is 8; or m is 3 and n is 7; or m is 4 and n is 6; or m is 6 and n is 4; or m is 7 and n is 3; or m is 8 and n is 2; or m is 9 and n is 1; or m is 10 and n is 0; or m is 0 and n is 11; or m is 1 and n is 10; or m is 2 and n is 9; or m is 3 and n is 8; or m is 4 and n is 7; or m is 5 and n is 6; or m is 6 and n is 5; or m is 7 and n is 4; or m is 8 and n is 3; or m is 9 and n is 2; or m is 10 and n is 1; or m is 11 and n is 0; or m is 0 and n is 12; or m is 1 and n is 11; or m is 2 and n is 10; or m is 3 and n is 9; or m is 4 and n is 8; or m is 5 and n is 7; or m is 7 and n is 5; or m is 8 and n is 4; or m is 9 and n is 3; or m is 10 and n is 2; or m is 11 and n is 1; or m is 12 and n is 0; or m is 0 and n is 13; or m is 1 and n is 12; or m is 2 and n is 11; or m is 3 and n is 10; or m is 4 and n is 9; or m is 5 and n is 8; or m is 6 and n is 7; or m is 7 and n is 6; or m is 8 and n is 5; or m is 9 and n is 4; or m is 10 and n is 3; or m is 11 and n is 2; or m is 12 and n is 1; or m is 13 and n is 0; or m is 0 and n is 14; or m is 1 and n is 13; or m is 2 and n is 12; or m is 3 and n is 11; or m is 4 and n is 10; or m is 5 and n is 9; or m is 6 and n is 8; or m is 8 and n is 6; or m is 9 and n is 5; or m is 10 and n is 4; or m is 11 and n is 3; or m is 12 and n is 2; or m is 13 and n is 1; or m is 14 and n is 0; or m is 0 and n is 15; or m is 1 and n is 14; or m is 2 and n is 13; or m is 3 and n is 12; or m is 4 and n is 11; or m is 5 and n is 10; or m is 6 and n is 9; or m is 7 and n is 8; or m is 8 and n is 7; or m is 9 and n is 6; or m is 10 and n is 5; or m is 11 and n is 4; or m is 12 and n is 3; or m is 13 and n is 2; or m is 14 and n is 1; or m is 15 and n is 0; or m is 0 and n is 16; or m is 1 and n is 15; or m is 2 and n is 14; or m is 3 and n is 13; or m is 4 and n is 12; or m is 5 and n is 11; or m is 6 and n is 10; or m is 7 and n is 9; or m is 9 and n is 7; or m is 10 and n is 6; or m is 11 and n is 5; or m is 12 and n is 4; or m is 13 and n is 3; or m is 14 and n is 2; or m is 15 and n is 1; or m is 16 and n is 0; or m is 1 and n is 16; or m is 2 and n is 15; or m is 3 and n is 14; or m is 4 and n is 13; or m is 5 and n is 12; or m is 6 and n is 11; or m is 7 and n is 10; or m is 8 and n is 9; or m is 9 and n is 8; or m is 10 and n is 7; or m is 11 and n is 6; or m is 12 and n is 5; or m is 13 and n is 4; or m is 14 and n is 3; or m is 15 and n is 2; or m is 16 and n is 1; or m is 17 and n is 0; or m is 0 and n is 18; or m is 1 and n is 17; or m is 2 and n is 16; or m is 3 and n is 15; or m is 4 and n is 14; or m is 5 and n is 13; or m is 6 and n is 12; or m is 7 and n is 11; or m is 8 and n is 10; or m is 10 and n is 8; or m is 11 and n is 7; or m is 12 and n is 6; or m is 13 and n is 5; or m is 14 and n is 4; or m is 15 and n is 3; or m is 16 and n is 2; or m is 17 and n is 1; or m is 18 and n is 0

Accordingly, some embodiments of the disclosure provide methods for preparing an olefinic alcohol product as described above, wherein the olefinic substrate is a metathesis product, and wherein the method includes: a) cross-metathesizing a first terminal olefin and a second different terminal olefin in the presence of a metathesis catalyst to form the metathesis product; and b) incubating the metathesis product with an enzyme capable of selectively hydroxylating one terminal carbon of the metathesis product to form an olefinic alcohol product.

In some embodiments, the first terminal olefin has the formula (CH₂═CH)(CH₂)_(m)H, the second different terminal olefin has the formula (CH₂═CH)(CH₂)_(n)H, the metathesis product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(n)H, the olefinic alcohol product has the formula HO(CH₂)_(m)(CH═CH)(CH₂)_(n)H, and m and n are different integers between 1 and 18. In some embodiments, the olefinic alcohol product has a chemical structure corresponding to an insect pheromone. In other embodiments, the olefinic alcohol product has a chemical structure corresponding to a precursor of a pheromone, wherein the precursor undergoes subsequent synthetic transformation, e.g., oxidation and/or acetylation, to produce a synthetically derived pheromone. In some embodiments, m and n are different integers between 1 and 9.

In some embodiments, methods described herein can be used to synthetically derive an olefinic alcohol which is an isomer of the olefinic alcohol product having a chemical structure corresponding to a pheromone or to a precursor of a pheromone. In some such embodiments, the pheromone isomer can be used in a pheromone composition.

In some embodiments, the olefinic alcohol product is a positional isomer of a pheromone which results from biohydroxylation of the terminal carbon on the n-end of the metathesis product, wherein the isomeric olefinic alcohol product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(n)OH, and m and n are different integers between 1 and 18. In some embodiments, m and n are different integers between 1 and 9. In other embodiments, the isomeric olefinic alcohol product can undergo a subsequent synthetic transformation, e.g., oxidation and/or acetylation, to produce a positional isomer of a pheromone.

In some embodiments, the olefinic alcohol product is a diol which results from biohydroxylation of the terminal carbon on the n-end and biohydroxylation of the terminal carbon on the m-end, the isomeric olefinic diol product has the formula HO(CH₂)_(m)(CH═CH)(CH₂)_(n)OH, and m and n are different integers between 1 and 18. In some embodiments, m and n are different integers between 1 and 9. In other embodiments, the olefinic diol product can undergo a subsequent synthetic transformation, e.g., oxidation and/or acetylation, to produce an analogue of a pheromone.

In some embodiments, the olefinic alcohol product is a positional isomer of a pheromone which results from biohydroxylation of a subterminal carbon on m-end of the metathesis produced, wherein the isomeric olefinic alcohol has the formula H(CH₂)_(i)CHOH(CH₂)_(m-i-1)(CH═CH)(CH₂)_(n)H. In some embodiments, the olefinic alcohol product is a positional isomer of a pheromone which results from biohydroxylation of a subterminal carbon on n-end of the metathesis produced, wherein the isomeric olefinic alcohol has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(n-i-1)CHOH(CH₂)H. In some embodiment, m, n and i are different integers between 1 and 17. In some embodiments, m, n, and i are different integers between 1 and 9.

The methods of the disclosure can also be conducted such that the biohydroxylation step is conducted prior to the metathesis step and/or other synthetic transformation steps. Accordingly, some embodiments of the disclosure provide methods wherein the olefinic substrate is a first terminal olefin, and wherein the method includes: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating the terminal carbon of the terminal olefin to form an α,ω-alkenol; and b) metathesizing the α,ω-alkenol and a second terminal olefin in the presence of a metathesis catalyst to form the olefinic alcohol product.

The alcohol can be protected with a suitable protecting group if necessary. In some embodiments, the methods of the disclosure include: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating the terminal carbon of the terminal olefin to form an α,ω-alkenol; b) protecting the α,ω-alkenol to form a protected α,ω-alkenol; c) metathesizing the protected α,ω-alkenol and a second terminal olefin in the presence of a metathesis catalyst to form a protected olefinic alcohol product; and d) deprotecting the protected olefinic alcohol product to form the olefinic alcohol product.

Any suitable alcohol protecting group can be used in the methods of the disclosure. Such protecting groups are well known to one of ordinary skill in the art, including those that are disclosed in Protective Groups in Organic Synthesis, 4th edition, T. W. Greene and P. G. M. Wuts, John Wiley & Sons, New York, 2006, which is incorporated herein by reference in its entirety. In some embodiments, the α,ω-alkenol is protected via esterification and the protected olefinic alcohol product is deprotected via hydrolysis. In some embodiments, the α,ω-alkenol is protected via esterification with an acid selected from the group consisting of formate and acetate.

Any suitable olefinic substrate can be used in methods where the biohydroxylation step is conducted prior to the metathesis step and/or other synthetic transformation steps. In some embodiments, the first terminal olefin has the formula (CH₂═CH)(CH₂)_(m)H, the α,ω-alkenol has the formula (CH₂═CH)(CH₂)_(m)OH, the second terminal olefin has the formula (CH₂═CH)(CH₂)_(n)H, the olefinic alcohol product has the formula H(CH₂)_(n)(CH═CH)(CH₂)_(m)OH, and m and n are each independently selected from an integer between 1 and 17. In some embodiments, m and n are each independently selected from an integer between 1 and 9.

Hydroxylation of Asymmetric Alkenes Using Alkyne Starting Material

In some embodiments, the alkene is produced according to Scheme 4 (see, Oprean et al. (2006) for the acetylation step and Buck and Chong (2001) for the alkyne alkylation step), Scheme 5 (see, Buck and Chong (2001) regarding the alkyne alkylation step), Scheme 6a, or Scheme 6b. Scheme 6b shows Wittig reaction conditions that favor the formation of the Z-isomer according to Smith et al. (2000).

Accordingly, some embodiments of the disclosure provide a method for synthesizing an olefinic alcohol product wherein the method includes:

a) forming a reaction mixture comprising a terminal alkyne according to formula I

wherein n is an integer from 0 to 16,

and an alkyl halide according to formula II

wherein X is a halogen and m is an integer from 0 to 16,

under conditions sufficient to form a disubstituted alkyne according to formula III

b) reducing the disubstituted alkyne to form an olefin according to formula IVa or IVb

and

c) incubating the olefin with an enzyme capable of selectively hydroxylating one terminal carbon of the olefin to form the olefinic alcohol product.

The terminal alkyne, the alkyl halide, the disubstituted alkyne, the olefin, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

In some embodiments, the disclosure includes:

a) forming a reaction mixture comprising a phosphonium salt according to formula XVI

wherein

-   -   each R is independently selected from C₁₋₆ alkyl and C₆₋₁₀ aryl,     -   X is a halogen, and     -   n is an integer from 0 to 16,

and an aldehyde according to formula XVII

wherein m is an integer from 0 to 16,

under conditions sufficient to form an olefin according to formula XVIIIa or formula XVIIIb

and

b) incubating the olefin with an enzyme capable of selectively hydroxylating one terminal carbon of the olefin to form the olefinic alcohol product.

The phosphonium salt, the aldehyde, the olefin, and the olefinic alcohol product can have any suitable combination of subscripts m and n, as described above. In some embodiments, m and n are independently selected integers between 1 and 9. In some embodiments, m and n are different integers between 1 and 9.

Metathesis Catalysts

In general, any metathesis catalyst stable under the reaction conditions and nonreactive with the functional groups present on the reactant shown in Schemes 3-6 may be used with the present disclosure. Such catalysts are, for example, those described by Grubbs (Grubbs, R. H., “Synthesis of large and small molecules using olefin metathesis catalysts.” PMSE Prepr., 2012), herein incorporated by reference in its entirety. Depending on the desired isomer of the olefin, as cis-selective metathesis catalyst may be used, for example one of those described by Shahane et al. (Shahane, S., et al. ChemCatChem, 2013. 5(12): p. 3436-3459), herein incorporated by reference in its entirety. Specific catalysts 1-5 exhibiting cis-selectivity are shown below (Scheme 7) and have been described previously (Khan, R. K., et al. J. Am. Chem. Soc., 2013. 135(28): p. 10258-61; Hartung, J. et al. J. Am. Chem. Soc., 2013. 135(28): p. 10183-5.; Rosebrugh, L. E., et al. J. Am. Chem. Soc., 2013. 135(4): p. 1276-9.; Marx, V. M., et al. J. Am. Chem. Soc., 2013. 135(1): p. 94-7.; Herbert, M. B., et al. Angew. Chem. Int. Ed. Engl., 2013. 52(1): p. 310-4; Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(4): p. 2040-3.; Keitz, B. K., et al. J. Am. Chem. Soc., 2012. 134(1): p. 693-9.; Endo, K. et al. J. Am. Chem. Soc., 2011. 133(22): p. 8525-7).

Additional Z-selective catalysts are described in (Cannon and Grubbs 2013; Bronner et al. 2014; Hartung et al. 2014; Pribisko et al. 2014; Quigley and Grubbs 2014) and are herein incorporated by reference in their entirety. Due to their excellent stability and functional group tolerance, in some embodiments metathesis catalysts include, but are not limited to, neutral ruthenium or osmium metal carbene complexes that possess metal centers that are formally in the +2 oxidation state, have an electron count of 16, are penta-coordinated, and are of the general formula LL′AA′M=CRbRc or LL′AA′M=(C═)nCRbRc (Pederson and Grubbs 2002); wherein

-   -   M is ruthenium or osmium;     -   L and L′ are each independently any neutral electron donor         ligand and selected from phosphine, sulfonated phosphine,         phosphite, phosphinite, phosphonite, arsine, stibnite, ether,         amine, amide, imine, sulfoxide, carboxyl, nitrosyl, pyridine,         thioether, or heterocyclic carbenes; and     -   A and A′ are anionic ligands independently selected from         halogen, hydrogen, C₁-C₂₀ alkyl, aryl, C₁-C₂₀ alkoxide,         aryloxide, C₂-C₂₀ alkoxycarbonyl, arylcarboxylate, C₁-C₂₀         carboxylate, arylsulfonyl, C₁-C₂₀ alkylsulfonyl, C₁-C₂₀         alkylsulfinyl; each ligand optionally being substituted with         C₁-C₅ alkyl, halogen, C₁-C₅ alkoxy; or with a phenyl group that         is optionally substituted with halogen, C₁-C₅ alkyl, or C₁-C₅         alkoxy; and A and A′ together may optionally comprise a         bidentate ligand; and     -   R_(b) and R_(c) are independently selected from hydrogen, C₁-C₂₀         alkyl, aryl, C₁-C₂₀ carboxylate, C₁-C₂₀ alkoxy, aryloxy, C₁-C₂₀         alkoxycarbonyl, C₁-C₂₀ alkylthio, C₁-C₂₀ alkylsulfonyl and         C₁-C₂₀ alkylsulfinyl, each of R_(b) and R_(c) optionally         substituted with C₁-C₅ alkyl, halogen, C₁-C₅ alkoxy or with a         phenyl group that is optionally substituted with halogen, C₁-C₅         alkyl, or C₁-C₅ alkoxy.

Other metathesis catalysts such as “well defined catalysts” can also be used. Such catalysts include, but are not limited to, Schrock's molybdenum metathesis catalyst, 2,6-diisopropylphenylimido neophylidenemolybdenum (VI) bis(hexafluoro-t-butoxide), described by Grubbs et al. (Tetrahedron 1998, 54: 4413-4450) and Basset's tungsten metathesis catalyst described by Couturier, J. L. et al. (Angew. Chem. Int. Ed. Engl. 1992, 31: 628).

Catalysts useful in the methods of the disclosure also include those described by Peryshkov, et al. J. Am. Chem. Soc. 2011, 133: 20754-20757; Wang, et al. Angewandte Chemie, 2013, 52: 1939-1943; Yu, et al. J. Am. Chem. Soc., 2012, 134: 2788-2799; Halford. Chem. Eng. News, 2011, 89 (45): 11; Yu, et al. Nature, 2011, 479: 88-93; Lee. Nature, 2011, 471: 452-453; Meek, et al. Nature, 2011: 471, 461-466; Flook, et al. J. Am. Chem. Soc. 2011, 133: 1784-1786; Zhao, et al. Org Lett., 2011, 13(4): 784-787; Ondi, et al. “High activity, stabilized formulations, efficient synthesis and industrial use of Mo- and W-based metathesis catalysts” XiMo Technology Updates, 2015: http://www.ximo-inc.com/files/ximo/uploads/download/Summary_3.11.15.pdf; Schrock, et al. Macromolecules, 2010: 43, 7515-7522; Peryshkov, et al. Organometallics 2013: 32, 5256-5259; Gerber, et al. Organometallics 2013: 32, 5573-5580; Marinescu, et al. Organometallics 2012: 31, 6336-6343; Wang, et al. Angew. Chem. Int. Ed. 2013: 52, 1939-1943; Wang, et al. Chem. Eur. 1 2013: 19, 2726-2740; and Townsend et al. J. Am. Chem. Soc. 2012: 134, 11334-11337.

Catalysts useful in the methods of the disclosure also include those described in International Pub. No. WO 2014/155185; International Pub. No. WO 2014/172534; U.S. Pat. Appl. Pub. No. 2014/0330018; International Pub. No. WO 2015/003815; and International Pub. No. WO 2015/003814.

Catalysts useful in the methods of the disclosure also include those described in U.S. Pat. No. 4,231,947; U.S. Pat. No. 4,245,131; U.S. Pat. No. 4,427,595; U.S. Pat. No. 4,681,956; U.S. Pat. No. 4,727,215; International Pub. No. WO 1991/009825; U.S. Pat. No. 5,087,710; U.S. Pat. No. 5,142,073; U.S. Pat. No. 5,146,033; International Pub. No. WO 1992/019631; U.S. Pat. No. 6,121,473; U.S. Pat. No. 6,346,652; U.S. Pat. No. 8,987,531; U.S. Pat. Appl. Pub. No. 2008/0119678; International Pub. No. WO 2008/066754; International Pub. No. WO 2009/094201; U.S. Pat. Appl. Pub. No. 2011/0015430; U.S. Pat. Appl. Pub. No. 2011/0065915; U.S. Pat. Appl. Pub. No. 2011/0077421; International Pub. No. WO 2011/040963; International Pub. No. WO 2011/097642; U.S. Pat. Appl. Pub. No. 2011/0237815; U.S. Pat. Appl. Pub. No. 2012/0302710; International Pub. No. WO 2012/167171; U.S. Pat. Appl. Pub. No. 2012/0323000; U.S. Pat. Appl. Pub. No. 2013/0116434; International Pub. No. WO 2013/070725; U.S. Pat. Appl. Pub. No. 2013/0274482; U.S. Pat. Appl. Pub. No. 2013/0281706; International Pub. No. WO 2014/139679; International Pub. No. WO 2014/169014; U.S. Pat. Appl. Pub. No. 2014/0330018; and U.S. Pat. Appl. Pub. No. 2014/0378637.

Catalysts useful in the methods of the disclosure also include those described in International Pub. No. WO 2007/075427; U.S. Pat. Appl. Pub. No. 2007/0282148; International Pub. No. WO 2009/126831; International Pub. No. WO 2011/069134; U.S. Pat. Appl. Pub. No. 2012/0123133; U.S. Pat. Appl. Pub. No. 2013/0261312; U.S. Pat. Appl. Pub. No. 2013/0296511; International Pub. No. WO 2014/134333; and U.S. Pat. Appl. Pub. No. 2015/0018557.

Catalysts useful in the methods of the disclosure also include those set forth in the following table:

Structure Name

dichloro[1,3-bis(2,6-isopropylphenyl)-2- imidazolidinylidene](benzylidene)(tricyclohex ylphosphine)ruthenium(II)

dichloro[1,3-bis(2,6-isopropylphenyl)-2- imidazolidinylidene](2- isopropoxyphenylmethylene)ruthenium(II)

dichloro[1,3-Bis(2-methylphenyl)-2- imidazolidinylidene](benzylidene)(tricyclohex ylphosphine)ruthenium(II)

dichloro[1,3-bis(2-methylphenyl)-2- imidazolidinylidene](2- isopropoxyphenylmethylene)ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene](benzylidene)bis(3- bromopyridine)ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene](3-methyl-2- butenylidene) (tricyclohexylphosphine) ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene][3-(2-pyridinyl) propylidene]ruthenium(II)

dichloro[1,3-bis(2,4,6-trimethylphenyl)-2- imidazolidinylidene][(tricyclohexylphosphora nyl)methylidene]ruthenium(II) tetrafluoroborate

dichloro(3-methyl-2-butenylidene) bis(tricyclohexylphosphine)ruthenium(II)

dichloro(3-methyl-2-butenylidene) bis(tricyclopentylphosphine)ruthenium(II)

dichloro(tricyclohexylphosphine)[(tricyclohexyl- phosphoranyl)methylidenelruthenium(II) tetrafluoroborate

bis(tricyclohexylphosphine) benzylidine ruthenium(IV) dichloride

[1,3-bis-(2,4,6-trimethylphenyl)-2- imidazolidinylidene]dichloro(phenylmethylene) (tricyclohexylphosphine)ruthenium

(1,3-bis-(2,4,6-trimethylphenyl)-2- imidazolidinylidene)dichloro(o- isopropoxyphenylmethylene)ruthenium

dichloro(o- isopropoxyphenylmethylene)(tricyclohexyl- phosphine)ruthenium(II)

[2-(1-methylethoxy-O)phenylmethyl- C](nitrato-O,O′){rel-(2R,5R,7R)-adamantane- 2,1-diyl[3-(2,4,6-trimethylphenyl)-1- imidazolidinyl-2-ylidene]}ruthenium

Catalysts useful in the methods of the disclosure also include those described in U.S. Pat. Appl. Pub. No. 2008/0009598; U.S. Pat. Appl. Pub. No. 2008/0207911; U.S. Pat. Appl. Pub. No. 2008/0275247; U.S. Pat. Appl. Pub. No. 2011/0040099; U.S. Pat. Appl. Pub. No. 2011/0282068; and U.S. Pat. Appl. Pub. No. 2015/0038723.

Catalysts useful in the methods of the disclosure include those described in International Pub. No. WO 2007/140954; U.S. Pat. Appl. Pub. No. 2008/0221345; International Pub. No. WO 2010/037550; U.S. Pat. Appl. Pub. No. 2010/0087644; U.S. Pat. Appl. Pub. No. 2010/0113795; U.S. Pat. Appl. Pub. No. 2010/0174068; International Pub. No. WO 2011/091980; International Pub. No. WO 2012/168183; U.S. Pat. Appl. Pub. No. 2013/0079515; U.S. Pat. Appl. Pub. No. 2013/0144060; U.S. Pat. Appl. Pub. No. 2013/0211096; International Pub. No. WO 2013/135776; International Pub. No. WO 2014/001291; International Pub. No. WO 2014/067767; U.S. Pat. Appl. Pub. No. 2014/0171607; and U.S. Pat. Appl. Pub. No. 2015/0045558.

The catalyst is typically provided in the reaction mixture in a sub-stoichiometric amount (e.g., catalytic amount). In certain embodiments, that amount is in the range of about 0.001 to about 50 mol % with respect to the limiting reagent of the chemical reaction, depending upon which reagent is in stoichiometric excess. In some embodiments, the catalyst is present in less than or equal to about 40 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than or equal to about 30 mol % relative to the limiting reagent. In some embodiments, the catalyst is present in less than about 20 mol %, less than about 10 mol %, less than about 5 mol %, less than about 2.5 mol %, less than about 1 mol %, less than about 0.5 mol %, less than about 0.1 mol %, less than about 0.015 mol %, less than about 0.01 mol %, less than about 0.0015 mol %, or less, relative to the limiting reagent. In some embodiments, the catalyst is present in the range of about 2.5 mol % to about 5 mol %, relative to the limiting reagent. In some embodiments, the reaction mixture contains about 0.5 mol % catalyst. In the case where the molecular formula of the catalyst complex includes more than one metal, the amount of the catalyst complex used in the reaction may be adjusted accordingly.

In some cases, the methods described herein can be performed in the absence of solvent (e.g., neat). In some cases, the methods can include the use of one or more solvents. Examples of solvents that may be suitable for use in the disclosure include, but are not limited to, benzene, p-cresol, toluene, xylene, diethyl ether, glycol, diethyl ether, petroleum ether, hexane, cyclohexane, pentane, methylene chloride, chloroform, carbon tetrachloride, dioxane, tetrahydrofuran (THF), dimethyl sulfoxide, dimethylformamide, hexamethyl-phosphoric triamide, ethyl acetate, pyridine, triethylamine, picoline, and the like, as well as mixtures thereof. In some embodiments, the solvent is selected from benzene, toluene, pentane, methylene chloride, and THF. In certain embodiments, the solvent is benzene.

In some embodiments, the method is performed under reduced pressure. This may be advantageous in cases where a volatile byproduct, such as ethylene, may be produced during the course of the metathesis reaction. For example, removal of the ethylene byproduct from the reaction vessel may advantageously shift the equilibrium of the metathesis reaction towards formation of the desired product. In some embodiments, the method is performed at a pressure of about less than 760 torr. In some embodiments, the method is performed at a pressure of about less than 700 torr. In some embodiments, the method is performed at a pressure of about less than 650 torr. In some embodiments, the method is performed at a pressure of about less than 600 torr. In some embodiments, the method is performed at a pressure of about less than 550 torr. In some embodiments, the method is performed at a pressure of about less than 500 torr. In some embodiments, the method is performed at a pressure of about less than 450 torr. In some embodiments, the method is performed at a pressure of about less than 400 torr. In some embodiments, the method is performed at a pressure of about less than 350 torr. In some embodiments, the method is performed at a pressure of about less than 300 torr. In some embodiments, the method is performed at a pressure of about less than 250 torr. In some embodiments, the method is performed at a pressure of about less than 200 torr. In some embodiments, the method is performed at a pressure of about less than 150 torr. In some embodiments, the method is performed at a pressure of about less than 100 torr. In some embodiments, the method is performed at a pressure of about less than 90 torr. In some embodiments, the method is performed at a pressure of about less than 80 torr. In some embodiments, the method is performed at a pressure of about less than 70 torr. In some embodiments, the method is performed at a pressure of about less than 60 torr. In some embodiments, the method is performed at a pressure of about less than 50 torr. In some embodiments, the method is performed at a pressure of about less than 40 torr. In some embodiments, the method is performed at a pressure of about less than 30 torr. In some embodiments, the method is performed at a pressure of about less than 20 torr. In some embodiments, the method is performed at a pressure of about 20 torr.

In some embodiments, the method is performed at a pressure of about 19 torr. In some embodiments, the method is performed at a pressure of about 18 torr. In some embodiments, the method is performed at a pressure of about 17 torr. In some embodiments, the method is performed at a pressure of about 16 torr. In some embodiments, the method is performed at a pressure of about 15 torr. In some embodiments, the method is performed at a pressure of about 14 torr. In some embodiments, the method is performed at a pressure of about 13 torr. In some embodiments, the method is performed at a pressure of about 12 torr. In some embodiments, the method is performed at a pressure of about 11 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 10 torr. In some embodiments, the method is performed at a pressure of about 9 torr. In some embodiments, the method is performed at a pressure of about 8 torr. In some embodiments, the method is performed at a pressure of about 7 torr. In some embodiments, the method is performed at a pressure of about 6 torr. In some embodiments, the method is performed at a pressure of about 5 torr. In some embodiments, the method is performed at a pressure of about 4 torr. In some embodiments, the method is performed at a pressure of about 3 torr. In some embodiments, the method is performed at a pressure of about 2 torr. In some embodiments, the method is performed at a pressure of about 1 torr. In some embodiments, the method is performed at a pressure of less than about 1 torr.

In some embodiments, the two metathesis reactants are present in equimolar amounts. In some embodiments, the two metathesis reactants are not present in equimolar amounts. In certain embodiments, the two reactants are present in a molar ratio of about 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In certain embodiments, the two reactants are present in a molar ratio of about 10:1. In certain embodiments, the two reactants are present in a molar ratio of about 7:1. In certain embodiments, the two reactants are present in a molar ratio of about 5:1. In certain embodiments, the two reactants are present in a molar ratio of about 2:1. In certain embodiments, the two reactants are present in a molar ratio of about 1:10. In certain embodiments, the two reactants are present in a molar ratio of about 1:7. In certain embodiments, the two reactants are present in a molar ratio of about 1:5. In certain embodiments, the two reactants are present in a molar ratio of about 1:2.

In general, the reactions with many of the metathesis catalysts disclosed herein provide yields better than 15%, better than 50%, better than 75%, or better than 90%. In addition, the reactants and products are chosen to provide at least a 5° C. difference, a greater than 20° C. difference, or a greater than 40° C. difference in boiling points. Additionally, the use of metathesis catalysts allows for much faster product formation than byproduct, it is desirable to run these reactions as quickly as practical. In particular, the reactions are performed in less than about 24 hours, less than 12 hours, less than 8 hours, or less than 4 hours.

One of skill in the art will appreciate that the time, temperature and solvent can depend on each other, and that changing one can require changing the others to prepare the pyrethroid products and intermediates in the methods of the disclosure. The metathesis steps can proceed at a variety of temperatures and times. In general, reactions in the methods of the disclosure are conducted using reaction times of several minutes to several days. For example, reaction times of from about 12 hours to about 7 days can be used. In some embodiments, reaction times of 1-5 days can be used. In some embodiments, reaction times of from about 10 minutes to about 10 hours can be used. In general, reactions in the methods of the disclosure are conducted at a temperature of from about 0° C. to about 200° C. For example, reactions can be conducted at 15-100° C. In some embodiments, reaction can be conducted at 20-80° C. In some embodiments, reactions can be conducted at 100-150° C.

Biohydroxylation to Produce a Pheromone and its Positional Isomer

As discussed above, an unsaturated hydrocarbon substrate can be subjected to biohydroxylation via an enzyme catalyst to thereby generate a mixture of a pheromone having a structure of an insect pheromone produced by a female insect and a positional isomer of the pheromone, which is not naturally produced by the female insect. The mixture of a pheromone and a positional isomer occurs through the enzyme catalyzing the hydrolysis of different carbons on the substrate, as shown in FIG. 1. In some embodiments, enzymes can be selected which catalyze hydroxylation of terminal carbons to produce unsaturated hydrocarbon products at the desired ratios. In some embodiments, an enzyme can be engineered to generate a mixture of unsaturated hydrocarbon products at desired ratios. Thus, in some embodiments, the ratio of the natural sex pheromone to the positional isomer can varied by selecting and/or engineering the biohydroxylation catalyst. In other embodiments, an enzyme can be selected and/or engineered to catalyze hydroxylation of an subterminal carbon.

Biohydroxylation Catalysts

Various enzymes and/or whole cells comprising enzymes can be used to catalyze hydroxylation reactions described above.

Known enzyme families with terminal hydroxylation activity for medium and long chain alkanes and fatty acids include AlkB, CYP52, CYP153, and LadA (Bordeaux et al., 2012, Angew. Chem.-Int. Edit. 51: 10712-10723; Ji et al., 2013, Front. Microbiol. 4). For example, Malca et al. describe terminal hydroxylation of mono-unsaturated fatty acid by cytochromes P450 of the CYP153 family (Malca et al., 2012, Chemical Communications 48: 5115-5117). Weissbart et al. describe the terminal hydroxylation of various cis and trans unsaturated lauric acid analogs (Weissbart et al., 1992, Biochimica et Biophysica Acta, Lipids and Lipid Metabolism 1124: 135-142). However, to date, none of these enzymes has been demonstrated to perform terminal hydroxylation of alkenes with internal olefins such as (E)-dec-5-ene. The presence of C═C bonds present competing sites of oxygen insertion and alters the 3-dimensional orientation of the molecule. The regioselectivity of these enzymes for the terminal C—H bond of alkanes and fatty acid substrate may not extend to alkenes with internal olefins for these reasons. For asymmetric substrates, obtaining hydroxylation at the desired terminal C—H bond presents additional challenges compared to symmetric substrates. Finally, controlling the reaction selectivity to produce a single terminal alcohol instead of α-ω diols, acids, or diacids is also a major concern.

In particular embodiments, the search for a terminal hydroxylase with activity for alkene with internal olefins starts with known terminal alkane and fatty acid hydroxylases. There are four families of enzymes with reported terminal alkane and fatty acid hydroxylation activity: (1) methane monooxygenases; (2) integral membrane diiron non-heme alkane hydroxylases (AlkB); (3) Cytochrome P450s (P450s); and (4) long chain alkane monooxygenases (LadA) (Bordeaux et al., 2012, Angew. Chem.-Int. Edit. 51: 10712-10723; Ji et al., 2013, Front. Microbiol. 4). Methane monooxygenases are difficult to express in heterologous non-methanotrophic hosts and generally prefer small substrate (<C4). Of the remaining three families, the substrate specificity based on substrate chain length of representative members is summarized below in Table 3.

TABLE 3 Relative activities of terminal hydroxylases for alkanes and fatty acids with various chain lengths. CYP153A1 AlkB CYP153A 6 (Scheps et CYP153A P. putida GPo1 alkB2 Gordonia 6 (Funhoff al., 2011, Org. P. sp. (Scheps (Vanbeilen et al., sp TF6* (Fujii et et al., 2006, Biomol. et al., 2011, 1994, Enzyme al., 2004, Biosci. Alkane/FA chain J. Bacteriol. Chem. 9: Org. Biomol. Microb. Technol. Biotechnol. length 188: 5220-5227) 6727-6733) Chem. 9: 6727-6733) 16: 904-911) Biochem. 68: 2171-2177) C8 100 100 100 95 72 C9 82 29 69 100 63 C10 23 13 60 60 66 C11 1 <8 <6 6 48 C12 34 C12 FA (lauric) C14 C14 FA (Myristic) C15 C16 C16 FA (Palmetic) C18 C18 FA (Stearic) C22 C24 CYP52 CYP52 CYP52A3 A4 (Scheller et A21 (Kim et al., LadA (Feng et al., (Scheller et al., al., 1996, Arch. 2007, Arch. 2007, Proc. Natl. 1996, Arch. Biochem Biochem. Alkane/FA chain Acad. Sci. U.S.A. Biochem Biophys. Biophys. 328: Biophys. 464: length 104: 5602-5607) 328: 245-254) 245-254) 213-220) C8 C9 C10 C11 C12 41 37 C12 FA (lauric) 20 100 100 C14 C14 FA 86 (Myristic) C15 83 C16 100 100 33 C16 FA 35 18 29 (Palmetic) C18 78 48 20 C18 FA 30 1 (Stearic) C22 74 C24 65 *100% relative activity obtained with hexane

In certain embodiments, depending on the chain length of the desired substrate, some members of these four enzyme families are better suited than others as candidates for evaluation. For C-10 substrates such as (E)-dec-5-ene, the substrate specificity of characterized CYP153 and AlkB enzymes makes them candidate enzymes. Likewise, for longer substrates such as (Z)-hexadec-11-ene, members of the LadA and CYP52 families appear to have the closest substrate profile.

The most widely characterized member of the AlkB family is obtained from the Alk system of Pseudomonas putida GPo1 (van Beilen and Funhoff, 2005, Curr. Opin. Biotechnol. 16: 308-314). In addition to the integral membrane diiron non-heme hydroxylase AlkB, a rubredoxin (AlkG) and a rubredoxin reductase (AlkT) are required for hydroxylation function. The entire Alk system of P. putida GPo1, alkBFGHJKL and alkST genes, which allows the strain to grown on alkanes as its sole carbon source, has been cloned into the broad host range vector pLAFR1 (pGEc47) and is available from DSMZ in the host E. Coli K12 Gec137 (Smits et al., 2001, Plasmid 46: 16-24). The other alk genes alkF, alkJ, alkH, alkK, alkL, and alkS encode an inactive rubredoxin, an alcohol dehydrogenase, an aldehyde dehydrogenase, an acyl-CoA synthase, an alkane transporter and a global pathway regulator, respectively (Smits et al., 2003, Antonie Van Leeuwenhoek 84: 193-200). These genes facilitate the use of the alcohol product from the AlkB reaction to generate the fatty acyl-CoA that is substrate for β-oxidation. To accumulate the alcohol product, a knockout strain of alkJ, E. coli GEC137 pGEc47ΔJ has been used in a whole-cell biotransformation to produce 1-dodecanol (Grant et al., 2011, Enzyme Microb. Technol. 48: 480-486). The presence of alkL appears to enhance substrate uptake and consequently improve the whole-cell activity for both Pseudomonas and E. coli (Cornelissen et al., 2013, Biotechnology and Bioengineering 110: 1282-1292; Julsing et al., 2012, Appl. Environ. Microbiol. 78: 5724-5733; Scheps et al., 2013, Microb. Biotechnol. 6: 694-707). A simplified version of pGEc47 containing only alkBFGST in the broad-host range vector pCOM10, pBT10, has also been used for the conversion of fatty-acid methyl esters to w-hydroxy fatty acid methyl esters in E. coli W3110 (Schrewe et al., 2011, Advanced Synthesis & Catalysis 353: 3485-3495).

CYP52 family members are membrane bound cytochrome P450s that require electron delivery from a reductase for function. CYP52 members have mainly been identified from alkane-degrading Candida species (Scheller et al., 1996, Arch. Biochem. Biophys. 328: 245-254; Craft et al., 2003, Appl. Environ. Microbiol. 69: 5983-5991; Scheller et al., 1998, J. Biol. Chem. 273: 32528-32534; Seghezzi et al., 1992, DNA Cell Biol. 11: 767-780; Zimmer et al., 1996, Biochem. Biophys. Res. Commun. 224: 784-789). Thus far, expression and characterization of CYP52 enzymes have been performed in the native Candida host and other yeast hosts. Gene knockouts of (1) the β-oxidation pathways, (2) alcohol dehydrogenases and (3) select native CYP52s has resulted in strains that can accumulate ω-hydroxy fatty acids when fatty acids are fed to the culture (Lu et al., 2010, J. Am. Chem. Soc. 132: 15451-15455). Of particular interest, DP428, DP522 and DP526 are C. tropicalis strains expressing a single CYP52 with the appropriate knockouts for catalyzing terminal hydroxylation of fatty acids (Lu et al., 2010, J. Am. Chem. Soc. 132: 15451-15455).

CYP153 family members are soluble and membrane associated cytochrome P450s that also depend on electron transfer from ferredoxin and ferredoxin reductase for function (Funhoff et al., 2007, Enzyme and Microbial Technology 40: 806-812). CYP153 members have been isolated from a range of alkane-degrading microorganisms. There are currently 56 annotated CYP153 sequences available from the Nelson P450 database, a BLAST search of CYP153A6 resulted in 221 identified homologs with >70% sequence identity. The use of CYP153 enzymes for terminal hydroxylation of octane and dodecanoic acid has been demonstrated with heterologous expression in E. coli. For the conversion of octane to octanol, the CYP153 operon from Mycobacterium sp. HXN-1500 was cloned into pET28b(+) and the biotransformation was performed in E. coli BL21(DE3) (Gudiminchi et al., 2012, Appl. Microbiol. Biotechnol. 96: 1507-1516). For the conversion of dodecanoic acid, an E. coli HMS174 strain containing a fusion of a CYP153A_(M.aq.) mutant with the CYP102A1 reductase domain in pColaDuet-1 along with alkL was used for the transformation (Scheps et al., 2013, Microb. Biotechnol. 6: 694-707).

Long chain alkane monooxygenase, LadA, isolated from G. thermodenitrificants NG80-2 catalyzes the terminal hydroxylation of C15 to C36 alkanes with a metal-free flavoprotein mechanism that differs from AlkB and CYP enzymes (Dong et al., 2012, Appl. Microbiol. Biotechnol. 94: 1019-1029). The LadA reaction requires FMNH₂ or NADPH and the native reductase partner has yet to be identified. Expression of the LadA gene in E. coli BL21 (DE3) using the pET-28a(+) plasmid yielded cell extracts with terminal hydroxylation activity for hexadecane (Dong et al., 2012, Appl. Microbiol. Biotechnol. 94: 1019-1029). Literature reports of LadA hydroxylation reactions have been performed using purified enzymes and examples of whole-cell biotransformation is lacking.

Coding sequences for enzymes that may be used herein may be derived from bacterial, fungal, or plant sources. Tables 3, 4, and 5 list enzymes for coding regions of representative non-heme diiron alkane monooxygenases, long-chain alkane hydroxylases, and cytochromes P450, respectively. Additional enzymes and their coding sequences may be identified by BLAST searching of public databases. Typically, BLAST searching of publicly available databases with known non-heme diiron alkane monooxygenases, cytochromes P450, and long-chain alkane hydroxylase sequences, such as those provided herein, is used to identify enzymes and their encoding sequences that may be used in the present disclosure. For example, enzymes having amino acid sequence identities of at least about 80-85%, 85%-90%, 90%-95%, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the enzymes listed in Tables 3, 4, and 5 may be used. Hydroxylase enzymes can be codon-optimized for expression in certain desirable host organisms, such as yeast and E. coli.

In other embodiments, the sequences of the enzymes provided herein may be used to identify other homologs in nature. For example, each of the encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, (1) methods of nucleic acid hybridization, (2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci. USA, 89:392 (1992)), and (3) methods of library construction and screening by complementation.

Hydroxylase enzymes or whole cells expressing hydroxylase enzymes can be further engineered for use in the methods of the disclosure. Enzymes can be engineered for improved hydroxylation activity, improved Z:E selectivity, improved regioselectivity, improved selectivity for hydroxylation over epoxidation and/or improved selectivity for hydroxylation over dehalogenation. The term “improved hydroxylation activity” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured in a comparable non-engineered hydroxylase enzyme of whole cells comprising a hydroxylase enzyme. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Mutations can be introduced into a hydroxylase enzyme resulting in engineered enzymes with improved hydroxylation activity. Methods to increase enzymatic activity are known to those skilled in the art. Such techniques can include increasing the expression of the enzyme by increasing plasmid copy number and/or use of a stronger promoter and/or use of activating riboswitches, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the KM for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003).

Accordingly, some embodiments of the disclosure provide methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is a non-heme diiron monooxygenase. In some embodiments, the non-heme diiron monooxygenase is selected from Table 4 or a variant thereof having at least 90% identity thereto.

TABLE 4 Non-heme diiron monooxygenase enzymes capable of catalyzing selective terminal alkene hydroxylation. Accession Species Origin Gene Name No Pseudomonas oleovorans alkB P12691 Pseudomonas mendocina (strain ymp) Pmen_0443 A4XPE8 Pseudomonas aeruginosa alkB Q932R7 Enterobacteriaceae bacterium 58 alkB B5TVB4 Bacillus sp. BTRH40 alkB B5TVB3 uncultured bacterium alkB B6Z2G6 Pseudomonas aeruginosa alk B7U6M1 uncultured bacterium alkB U3PXQ1 uncultured bacterium alkB U3Q1X4 Pseudomonas stutzeri (Pseudomonas alkB Q7X4G8 perfectomarina) uncultured organism alkB G3EBX4 uncultured bacterium alkB U3PXQ7 Pseudomonas aeruginosa alk B7U6M0 Pseudomonas chlororaphis subsp. alkB Q9RLI5 aureofaciens Arthrobacter sp. ITRH48 alkB B5TVB7 Streptomyces sp. ITRH51 alkB B5TVB6 Arthrobacter sp. ITRH49 alkB B5TVC0 Dietzia sp. ITRH56 alkB B5TVB8 Microbacterium sp. ITRH47 alkB B5TVB5 Pantoea sp. BTRH11 alkB B5TVB2 Pseudomonas sp. ITRI53 alkB B5TVB1 Pseudomonas sp. ITRI73 alkB B5TVB0 Pseudomonas sp. ITRH25 alkB B5TVA9 Pseudomonas sp. MIXRI75 alkB B5TVA8 Pseudomonas sp. MIXRI74 alkB B5TVA7 Rhodococcus sp. ITRH43 alkB B5TVA4 Ochrobactrum sp. ITRH1 alkB B5TVA3 Alcaligenaceae bacterium BTRH5 alkB B5TVA6 Pseudomonas sp. ITRH76 alkB B5TVA5 Pseudomonas sp. 7/156 alkB Q93LR8 uncultured Rhizobiales bacterium alkB D6NSH3 uncultured soil bacterium S5DSW0 uncultured bacterium alkB U3PYH2 uncultured prokaryote alkB C7EAT4 uncultured Rhizobiales bacterium alkB D6NSL1 uncultured Rhizobiales bacterium alkB D6NSJ4 uncultured prokaryote alkB C7EAZ5 uncultured Rhizobiales bacterium alkB D6NSK5 uncultured Rhizobiales bacterium alkB D6NSK3 uncultured Rhizobiales bacterium alkB D6NSJ7 uncultured Rhizobiales bacterium alkB D6NSK1 uncultured Rhizobiales bacterium alkB D6NSH4 uncultured Rhizobiales bacterium alkB D6NSJ2 uncultured Rhizobiales bacterium alkB D6NSI2 uncultured Rhizobiales bacterium alkB D6NSJ3 uncultured Rhizobiales bacterium alkB D6NSJ6 Pseudomonas sp. ITRI22 alkB B5TVB9 uncultured Rhizobiales bacterium alkB D6NSK7 uncultured soil bacterium S5DTG4 Pseudomonas putida (Arthrobacter alkB Q9WWW6 siderocapsulatus) uncultured Rhizobiales bacterium alkB D6NSI6 uncultured bacterium alkB B6Z2E6 uncultured bacterium alkB B1P6K4 Pseudomonas sp. G5(2012) PG5_40690 S2EW96 Alcanivorax dieselolei alkB B6Z2B7 Alcanivorax borkumensis alkB B6Z284 uncultured bacterium alkB B6Z2G9 Marinobacter sp. S17-4 alkB C7DLJ8 uncultured bacterium alkB B6Z2H0 Alcanivorax sp. S17-16 alkB B6Z2D8 uncultured organism alkB G3EBX7 uncultured bacterium alkB H9NJ23 uncultured bacterium alkB C8AYB7 uncultured bacterium alkB W0UB63 uncultured bacterium alkB U3Q1V0 Alcanivorax borkumensis alkB T1WPB9 uncultured organism alkB G3EBX5 uncultured Rhizobiales bacterium alkB D6NSK6 uncultured bacterium alkB U3Q5C8 uncultured bacterium alkB Q3HXE5 Xanthobacter flavus alkane-1-monooxygenase Q934J9 uncultured bacterium alkB Q3HXD6 Acidisphaera sp. C197 alkB Q5RLH8 uncultured bacterium alkB M9T624 uncultured bacterium alkB M9T8D1 uncultured bacterium alkB H9B8U8 Kordiimonas gwangyangensis alkB B6Z2E4 uncultured soil bacterium S5DPL2 uncultured bacterium alkB F0X332 uncultured bacterium alkB F0X324 uncultured bacterium alkB F0X334 uncultured organism alkB G3EBX2 uncultured bacterium alkB F0X328 uncultured soil bacterium S5DTI7 uncultured bacterium alkB Q3HXF7 uncultured bacterium alkB F0X327 uncultured bacterium alkB F0X335 uncultured bacterium alkB F0X329 uncultured bacterium alkB F0X342 uncultured bacterium alkB F0X300 uncultured bacterium alkB Q3HXE8 uncultured bacterium alkB U3Q1X0 uncultured bacterium alkB Q3HXD7 Ralstonia sp. PT11 alkB Q3HXC9 uncultured bacterium alkB Q3HXE6 uncultured bacterium alkB F0X305 uncultured bacterium alkB U3Q5A0 uncultured bacterium alkB F0X306 Marinobacter sp. P1-14D alkB1 C6KEH4 uncultured Rhizobiales bacterium alkB D6NSI7 uncultured bacterium alkB F0X346 uncultured bacterium alkB F0X346 uncultured bacterium alkB F0X343 uncultured bacterium alkB F0X339 uncultured bacterium alkB F0X309 uncultured bacterium alkB F0X333 uncultured bacterium alkB F0X321 uncultured bacterium alkB Q3HXF0 uncultured bacterium alkB F0X312 uncultured bacterium alkB F0X303 uncultured bacterium alkB F0X331 uncultured bacterium alkB F0X302 uncultured bacterium alkB Q3HXE9 uncultured bacterium alkB F0X313 uncultured bacterium alkB F0X316 uncultured bacterium alkB M9TDK6 uncultured bacterium alkB H9B8V5 uncultured Rhizobiales bacterium alkB D6NSF4 uncultured Rhizobiales bacterium alkB D6NSF2 uncultured bacterium alkB B6Z2G8 uncultured Rhizobiales bacterium alkB D6NSF1 uncultured Rhizobiales bacterium alkB D6NSG4 uncultured Rhizobiales bacterium alkB D6NSG3 uncultured Rhizobiales bacterium alkB D6NSF3 uncultured Rhizobiales bacterium alkB D6NSI4 uncultured Rhizobiales bacterium alkB D6NSH9 uncultured Rhizobiales bacterium alkB D6NSG1 uncultured Rhizobiales bacterium alkB D6NSJ9 uncultured Rhizobiales bacterium alkB D6NSG6 uncultured soil bacterium S5DP42 uncultured bacterium alkB F0X323 uncultured bacterium alkB F0X318 uncultured bacterium alkB F0X317 uncultured bacterium alkB F0X325 uncultured bacterium alkB F0X308 uncultured bacterium alkB F0X336 uncultured soil bacterium S5E0W0 uncultured bacterium alkB F0X304 Bradyrhizobium sp. DFCI-1 C207_00091 U1HQ84 uncultured Rhizobiales bacterium alkB D6NSF9 uncultured Rhizobiales bacterium alkB D6NSH2 uncultured Rhizobiales bacterium alkB D6NSF6 uncultured Rhizobiales bacterium alkB D6NSG2 uncultured Rhizobiales bacterium alkB D6NSH7 uncultured bacterium alkB F0X322 uncultured soil bacterium S5DPY4 uncultured bacterium alkB F0X349 uncultured bacterium alkB F0X310 uncultured bacterium alkB F0X315 uncultured bacterium alkB F0X344 uncultured bacterium alkB F0X326 uncultured bacterium alkB W0UB94 uncultured bacterium alkB W0UAL7 uncultured soil bacterium S5DP84 uncultured soil bacterium S5E064 uncultured soil bacterium S5E0M5 uncultured bacterium alkB M9T7Y4 uncultured prokaryote alkB C7EAZ7 Thalassolituus oleivorans alkB Q8RSS6 uncultured prokaryote alkB C7EAZ8 Marinobacter sp. EVN1 Q672_13115 U7NVU4 uncultured Rhizobiales bacterium alkB D6NSF8 Marinobacter aquaeolei (strain ATCC Maqu_0610 A1TY92 700491/DSM 11845/VT8) (Marinobacter hydrocarbonoclasticus (strain DSM 11845)) Marinobacter hydrocarbonoclasticus alkB MARHY2847 H8WCU7 ATCC 49840 uncultured Rhizobiales bacterium alkB D6NSG8 Alcanivorax borkumensis alkB1 Q93UQ1 Alcanivorax borkumensis (strain SK2/ alkB1 ABO_2707 Q0VKZ3 ATCC 700651/DSM 11573) Marinobacter aquaeolei (strain ATCC Maqu_0440 A1TXS2 700491/DSM 11845/VT8) (Marinobacter hydrocarbonoclasticus (strain DSM 11845)) Alcanivorax sp. 97CO-5 Y017_07510 W7AC06 Marinobacter sp. C1S70 Q667_13505 U7P171 Marinobacter sp. EVN1 Q672_13130 U7NYF9 Pseudoxanthomonas spadix (strain BD- DSC_08960 G7UVX3 a59) Marinobacter sp. EN3 Q673_04890 U7H9M7 Marinobacter sp. ES-1 Q666_09550 U7G9A6 Oceanicaulis sp. HTCC2633 OA2633_08724 A3UHL2 Citreicella sp. 357 C357_19621 I1AS33 Caulobacter sp. (strain K31) Caul_5439 B0TA04 Thalassolituus oleivorans MIL-1 TOL_1423 M5DQR5 uncultured bacterium alkB W0UAQ4 uncultured bacterium alkB W0UAL9 uncultured bacterium alkB W0UAQ9 gamma proteobacterium NOR5-3 NOR53_3428 B8KLY6 uncultured marine microorganism 21G8-5 A5CFX9 uncultured marine microorganism 9E7-8 A5CFU5 Alcanivorax pacificus W11-5 S7S_02132 K2GLA3 Alcanivorax dieselolei C3W4W7 Alcanivorax sp. PN-3 Q668_06955 U7I1M1 Alcanivorax dieselolei (strain DSM 16502/ B5T_00721 K0C8Z6 CGMCC 1.3690/B-5) Alcanivorax dieselolei alkB2 D2JNY2 bacterium enrichment culture clone US3- mdpA L7T214 MTBE bacterium enrichment culture clone US2- mdpA L7SZY0 MTBE Marinobacter sp. ELB17 MELB17_10558 A3JHB9 Marinobacter sp. BSs20148 alkB1 MRBBS_1602 M1FBW8 Pseudomonas alcaligenes NBRC 14159 alkB PA6_005_01830 U3AUD1 Simiduia agarivorans (strain DSM 21679/ M5M_18065 K4KP06 JCM 13881/BCRC 17597/SA1) gamma proteobacterium HTCC2207 GB2207_03060 Q1YPC4 Limnobacter sp. MED105 LMED105_14555 A6GTF8 Alcanivorax sp. R8-12 alkB2 R9R6I2 Gammaproteobacteria bacterium alkB1 U062_00014 W2UFM4 MOLA455 Alcanivorax hongdengensis A-11-3 A11A3_01150 L0WGR7 Acidovorax sp. KKS102 C380_12125 K0IAD8 Moritella sp. PE36 PE36_11657 A6FHH9 Moritella sp. PE36 PE36_11657 A6FHH9 Ahrensia sp. R2A130 alkB R2A130_3229 E0MP68 Hoeflea phototrophica DFL-43 HPDFL43_04645 A9D3P4 Curvibacter putative symbiont of Hydra alkB Csp_A02180 C9Y7W7 magnipapillata Pseudovibrio sp. JE062 PJE062_1512 B6QXF8 Oxalobacteraceae bacterium IMCC9480 IMCC9480_2292 F1W4Y4 Methylibium petroleiphilum (strain PM1) alkB Mpe_B0606 A2SP81 Ralstonia sp. AU12-08 C404_01360 S9TME3 Burkholderia phytofirmans (strain DSM Bphyt_5401 B2TBV7 17436/PsJN) gamma proteobacterium BDW918 DOK_05250 I2JMD3 Pseudovibrio sp. (strain FO-BEG1) alkB PSE_3490 G8PKM2 Bradyrhizobium sp. DFCI-1 C207_06028 U1H8I8 Alcanivorax dieselolei (strain DSM 16502/ B5T_04393 K0CLJ4 CGMCC 1.3690/B-5) Alcanivorax sp. PN-3 Q668_04650 U7HLN0 Alcanivorax dieselolei alkB1 Q6B431 Burkholderia thailandensis E444 BTJ_212 W6C501 Burkholderia thailandensis 2002721723 BTQ_2100 W6BLA1 Burkholderia thailandensis H0587 BTL_1506 W6BA85 Burkholderia thailandensis (strain E264/ BTH_I1814 Q2SXK3 ATCC 700388/DSM 13276/CIP 106301) Burkholderia pseudomallei 1026b BP1026B_I0975 I1WH83 Burkholderia pseudomallei 1026a BP1026A_4019 I2KNJ5 Burkholderia pseudomallei MSHR305 BDL_3139 S5P5X7 Burkholderia pseudomallei 305 alkB BURPS305_7408 A4LDP5 Burkholderia pseudomallei Pasteur 52237 alkB BURPSPAST_R0133 A8KVJ2 Burkholderia pseudomallei (strain BPSL2350 Q63SH1 K96243) Burkholderia pseudomallei (strain 1710b) BURPS1710b_2801 Q3JQG8 Burkholderia pseudomallei BPC006 BPC006_I2776 K7Q7Y2 Burkholderia pseudomallei 1710a alkB BURPS1710A_3234 C6TUD4 Burkholderia pseudomallei 1106b alkB_2BURPS1106B_A1957 C5ZKC8 Burkholderia pseudomallei (strain 1106a) alkB BURPS1106A_2735 A3NXB5 Burkholderia pseudomallei (strain 668) BURPS668_2678 A3NBI1 Burkholderia pseudomallei NCTC 13178 BBJ_481 V9Y591 Burkholderia pseudomallei MSHR1043 D512_14116 M7EHA3 Burkholderia pseudomallei 354a BP354A_0895 I2MQ94 Burkholderia pseudomallei 354e BP354E_0708 I2MD23 Burkholderia pseudomallei 1258b BP1258B_0905 I2LQQ4 Burkholderia pseudomallei 1258a BP1258A_0812 I2LKD3 Burkholderia pseudomallei 576 alkB BUC_2998 B7CM79 Burkholderia pseudomallei 1655 alkB BURPS1655_H0133 B2HAC8 Burkholderia pseudomallei S13 alkB BURPSS13_V0139 B1HDJ2 Burkholderia pseudomallei 406e alkB BURPS406E_H0229 A8EBS1 Burkholderia pseudomallei MSHR146 BBN_1088 W0PXC8 Burkholderia pseudomallei MSHR511 BBQ_961 W0MCN0 Burkholderia pseudomallei NAU20B-16 BBS_2570 V9YGA1 Burkholderia pseudomallei MSHR346 GBP346_A2857 C4KQU6 Burkholderia pseudomallei MSHR338 M218_13015 W1M8G5 Burkholderia xenovorans (strain LB400) Bxe_B1208 Q13ME1 Burkholderia thailandensis MSMB43 A33K_14899 I6AHY8 Burkholderia sp. Ch1-1 BCh11DRAFT_02054 I2IU52 Alcanivorax sp. R8-12 alkB3 R9R6Q8 gamma proteobacterium HTCC5015 GP5015_636 B5JV27 Alcanivorax pacificus W11-5 S7S_03034 K2GFU4 Actinoplanes sp. (strain ATCC 31044/ alkB ACPL_4910 G8SLX8 CBS 674.73/SE50/110) Alcanivorax sp. DG881 ADG881_1174 B4X426 Methylibium sp. T29-B alkB1 Y694_03823 W7WAG2 Methylibium sp. T29 mdpA X551_03232 W7VT91 Burkholderia thailandensis MSMB121 BTI_1284 N0AI18 Burkholderia sp. TJI49 B1M_44170 F0GKQ0 Burkholderia mallei (strain ATCC 23344) alkB BMA0635 Q62LK2 Burkholderia mallei (strain NCTC 10247) alkB BMA10247_1692 A3MLU7 Burkholderia mallei (strain NCTC 10229) alkB BMA10229_A2910 A2SA87 Burkholderia mallei (strain SAVP1) alkB BMASAVP1_A2377 A1V630 Burkholderia mallei PRL-20 alkB BMAPRL20_A0647 C5NLY3 Burkholderia mallei GB8 horse 4 BMAGB8_0674 C4AYJ3 Burkholderia mallei ATCC 10399 alkB BMA10399_E0136 A9KA35 Burkholderia mallei JHU alkB BMAJHU_C0140 A5XN41 Burkholderia mallei FMH alkB BMAFMH_C0136 A5XJ42 Burkholderia mallei 2002721280 alkB BMA721280_A1345 A5TJ65 Burkholderia pseudomallei Pakistan 9 alkB BUH_2787 C0YFB6 Burkholderia sp. (strain 383) (Burkholderia Bcep18194_A4085 Q39IN4 cepacia (strain ATCC 17760/NCIB 9086/ R18194)) Ralstonia sp. 5_2_56FAA HMPREF0989_00681 U3G9A8 Ralstonia sp. 5_7_47FAA HMPREF1004_00261 E2ST40 Burkholderia cenocepacia (strain AU Bcen_0501 Q1BY92 1054) Burkholderia cenocepacia (strain HI2424) Bcen2424_0980 A0K5F6 Burkholderia sp. KJ006 MYA_0870 I2DKR1 Burkholderia vietnamiensis (strain G4/ Bcep1808_0897 A4JCA5 LMG 22486) (Burkholderia cepacia (strain R1808)) Burkholderia cenocepacia KC-01 P355_2107 V5A0K9 Ralstonia pickettii (strain 12D) Rpic12D_4221 C6BN09 Ralstonia pickettii (strain 12J) Rpic_4109 B2UI09 Ralstonia pickettii OR214 OR214_00862 R0CSD0 Mycobacterium thermoresistibile ATCC KEK_22639 G7CND0 19527 Burkholderia cenocepacia PC184 BCPG_00786 A2VS55 Parvularcula bermudensis (strain ATCC PB2503_09204 E0TD71 BAA-594/HTCC2503/KCTC 12087) Rhodococcus triatomae BKS 15-14 G419_20650 M2WXQ1 Alcanivorax hongdengensis A-11-3 A11A3_01155 L0WH65 Alcanivorax hongdengensis G1C7G7 Micromonospora sp. ATCC 39149 MCAG_04553 C4REI2 Micromonospora lupini str. Lupac 08 alkB MILUP08_41795 I0KZ81 Patulibacter medicamentivorans PAI11_23570 H0E6A7 Burkholderia cenocepacia (strain ATCC BCAL3029 B4EBR3 BAA-245/DSM 16553/LMG 16656/ NCTC 13227/J2315/CF5610) (Burkholderia cepacia (strain J2315)) Burkholderia cenocepacia BC7 BURCENBC7_AP5666 U1ZCU6 Burkholderia cenocepacia K56-2Valvano BURCENK562V_C5856 T0E860 Burkholderia cenocepacia H111 I35_3695 G7HIJ0 Burkholderia cepacia GG4 GEM_2548 J7J4L5 Burkholderia ambifaria IOP40-10 BamIOP4010DRAFT_1629 B1FC70 Burkholderia vietnamiensis AU4i L810_3738 U2H0D0 Burkholderia ambifaria MEX-5 BamMEX5DRAFT_0109 B1SX43 Burkholderia cenocepacia (strain MC0-3) Bcenmc03_0941 B1JX99 Burkholderia cepacia (Pseudomonas alkB Q9AEN3 cepacia) Burkholderia multivorans CGD1 BURMUCGD1_2488 B9BAK1 Burkholderia multivorans (strain ATCC alkB BMULJ_00816 B3CYB3 17616/249) Burkholderia multivorans (strain ATCC alkB BMULJ_00816 B3CYB3 17616/249) Burkholderia multivorans CGD2M BURMUCGD2M_2894 B9CFY2 Burkholderia multivorans CGD2 BURMUCGD2_2807 B9BSN6 Burkholderia glumae (strain BGR1) bglu_1g25240 C5AA12 Burkholderia multivorans CF2 BURMUCF2_0698 J5AST2 Burkholderia multivorans ATCC BAA-247 BURMUCF1_0763 J4JJJ2 Mycobacterium xenopi RIVM700367 MXEN_06581 I0RWI2 Alcanivorax sp. P2S70 Q670_07625 U7G3V1 Rhodococcus sp. p52 alkB U5S015 Rhodococcus pyridinivorans AK37 AK37_15478 H0JTS8 Micromonospora sp. M42 MCBG_00051 W7V9N0 Nocardia nova SH22a NONO_c63170 W5TPA6 Actinoplanes missouriensis (strain ATCC AMIS_28610 I0H4Z4 14538/DSM 43046/CBS 188.64/JCM 3121/NCIMB 12654/NBRC 102363/ 431) Mycobacterium thermoresistibile ATCC KEK_04707 G7CD93 19527 Streptomyces collinus Tu 365 B446_00650 B446_34640 S5VEV9 Mycobacterium smegmatis MKD8 alkB D806_1894 L8FH78 Mycobacterium smegmatis (strain ATCC alkB MSMEG_1839 A0QTH1 700084/mc(2)155) MSMEI_1797 Burkholderia gladioli (strain BSR3) bgla_1g28520 F2LCU4 Nocardia cyriacigeorgica (strain GUH-2) alkB NOCYR_2725 H6R6Y1 Mycobacterium sp. (strain Spyr1) Mspyr1_40540 E6TPD9 Mycobacterium gilvum (strain PYR-GCK) Mflv_4721 A4TF88 (Mycobacterium flavescens (strain ATCC 700033/PYR-GCK)) Mycobacterium hassiacum DSM 44199 C731_1322 K5BKD8 Mycobacterium phlei RIVM601174 MPHLEI_02293 I0S2Q3 Burkholderia ambifaria (strain MC40-6) BamMC406_0853 B1YUL7 Conexibacter woesei (strain DSM 14684/ Cwoe_5739 D3F1V9 JCM 11494/NBRC 100937/ID131577) Burkholderia ambifaria (strain ATCC Bamb_0841 Q0BHH3 BAA-244/AMMD) (Burkholderia cepacia (strain AMMD)) Mycobacterium vaccae ATCC 25954 MVAC_06502 K0V939 Streptomyces sp. AA4 SSMG_06597 D9UYP9 Nocardia asteroides NBRC 15531 alkB NCAST_33_00580 U5EK43 Hydrocarboniphaga effusa AP103 WQQ_35830 I8T3V4 Mycobacterium sp. (strain Spyr1) Mspyr1_27000 E6TM45 Rhodococcus sp. EsD8 EBESD8_14280 N1M251 Rhodococcus pyridinivorans SB3094 Y013_10875 Y013_14995 V9XCI1 uncultured bacterium alk A7XY59 Dietzia sp. D5 W0C8S6 Gordonia amarae NBRC 15530 alkB GOAMR_34_00200 G7GP29 gamma proteobacterium BDW918 DOK_15269 I2JH75 Marinobacter sp. EVN1 Q672_03155 U7NQ32 Marinobacter santoriniensis NKSG1 MSNKSG1_09613 M7CV98 Marinobacter sp. ES-1 Q666_05770 U7GFG6 gamma proteobacterium HdN1 alkM HDN1F_04190 E1VGR0 Nocardia farcinica (strain IFM 10152) NFA_33210 Q5YUH3 Mycobacterium chubuense (strain NBB4) Mycch_2783 I4BJT7 Acinetobacter towneri DSM 14962 = CIP F947_01315 N9CH84 107472 Rhodococcus erythropolis CCM2595 O5Y_10330 T1VNI2 Rhodococcus erythropolis (strain PR4/ alkB RER_21620 C0ZWY5 NBRC 100887) Rhodococcus sp. P27 N806_20680 U0E9X4 Rhodococcus erythropolis DN1 N601_09550 T5IBP7 Rhodococcus erythropolis (Arthrobacter alkB A4ZZL2 picolinophilus) Mycobacterium fortuitum subsp. fortuitum MFORT_07571 K0VIS2 DSM 46621 Rhodococcus qingshengii BKS 20-40 G418_14624 M2XAS5 Rhodococcus erythropolis (Arthrobacter alkB2 Q9AE68 picolinophilus) Rhodococcus sp. (strain RHA1) alkB RHA1_ro02534 Q0SDP7 Rhodococcus sp. JVH1 JVH1_3134 J1RMD5 Rhodococcus wratislaviensis IFP 2016 Rwratislav_18854 L2TK91 Rhodococcus wratislaviensis alkB1 K7WI49 Rhodococcus sp. (strain Q15) alkB2 Q93DM7 Rhodococcus opacus M213 WSS_A20069 K8XV97 Rhodococcus erythropolis (Arthrobacter alkB V5LET8 picolinophilus) Streptomyces sp. AA4 SSMG_06805 D9V1L5 Geobacillus sp. MH-1 alkB-geo6 C5J0F7 Mycobacterium neoaurum VKM Ac- D174_08465 V5X9E7 1815D Rhodococcus imtechensis RKJ300 = JCM W59_13161 I0WSJ7 13270 Prauserella rugosa alkB Q9XBM1 Rhodococcus erythropolis SK121 RHOER0001_4201 C3JG64 Amycolatopsis azurea DSM 43854 C791_5134 M2PZK0 Mycobacterium rhodesiae (strain NBB3) MycrhN_0412 G8RK27 Rhodococcus ruber alkB7 D3U111 Rhodococcus ruber BKS 20-38 G352_25762 M2XQQ3 Mycobacterium chubuense (strain NBB4) D2JYT1 Mycobacterium chubuense (strain NBB4) Mycch_1351 I4BFU6 Mycobacterium smegmatis JS623 Mycsm_01384 L0IUF4 Nocardia nova SH22a alkB NONO_c46180 W5TJL9 Rhodococcus sp. BCP1 alkB E5G6V9 Saccharomonospora marina XMU15 SacmaDRAFT_4417 H5X9W5 Mycobacterium sp. (strain JLS) Mjls_1369 A3PW94 Rhodococcus ruber alkB7 D3U119 Mycobacterium tuberculosis BT1 alkB HKBT1_3428 W6HJ76 Mycobacterium tuberculosis BT2 alkB HKBT2_3435 W6H3Z6 Mycobacterium tuberculosis HKBS1 alkB HKBS1_3438 W6GVB7 Mycobacterium tuberculosis EAI5 M943_16800 S5F023 Mycobacterium tuberculosis J114_17435 R4MLW1 EAI5/NITR206 Mycobacterium tuberculosis J113_22685 R4MIF7 CAS/NITR204 Mycobacterium bovis (strain ATCC BAA- alkB Mb3280c Q7TWW3 935/AF2122/97) Mycobacterium tuberculosis (strain ATCC alkB Rv3252c RVBD_3252c O05895 25618/H37Rv) Mycobacterium tuberculosis str. J112_17475 M9UX97 Beijing/NITR203 Mycobacterium bovis BCG str. Korea K60_033810 M1IQ04 1168P Mycobacterium liflandii (strain 128FXT) alkB MULP_01451 L7V4G7 Mycobacterium tuberculosis (strain CDC alkB MT3350 L7N540 1551/Oshkosh) Mycobacterium canettii CIPT 140070017 alkB BN45_60281 L0QZH1 Mycobacterium canettii CIPT 140070008 alkB BN43_60261 L0QC77 Mycobacterium canettii CIPT 140060008 alkB BN44_70036 L0Q026 Mycobacterium tuberculosis 7199-99 MT7199_3294 L0NZI4 Mycobacterium tuberculosis KZN 605 TBXG_003280 I6RJV1 Mycobacterium tuberculosis KZN 4207 TBSG_03323 I1SDS8 Mycobacterium tuberculosis RGTB327 MRGA327_20020 H8HLB9 Mycobacterium tuberculosis (strain ATCC alkB ERDMAN_3566 H8EY95 35801/TMC 107/Erdman) Mycobacterium tuberculosis UT205 alkB UDA_3252c H6S7Q5 Mycobacterium bovis BCG str. Mexico alkB BCGMEX_3279c G7QY42 Mycobacterium tuberculosis CTRI-2 alkB MTCTRI2_3319 G2N7Q9 Mycobacterium canettii (strain CIPT alkB MCAN_32711 G0THM9 140010059) Mycobacterium canettii (strain CIPT alkB MCAN_32711 G0THM9 140010059) Mycobacterium africanum (strain alkB MAF_32630 F8M6G6 GM041182) Mycobacterium tuberculosis (strain alkB CCDC5180_2963 F7WQM1 CCDC5180) CFBR_3446 Mycobacterium tuberculosis (strain alkB CCDC5079_3000 F7WLN9 CCDC5079) CFBS_3441 Mycobacterium tuberculosis (strain KZN TBMG_03300 C6DXJ8 1435/MDR) Mycobacterium bovis (strain BCG/Tokyo alkB JTY_3277 C1AH26 172/ATCC 35737/TMC 1019) Mycobacterium marinum (strain ATCC alkB MMAR_1291 B2HEP2 BAA-535/M) Mycobacterium tuberculosis (strain F11) TBFG_13281 A5WSG7 Mycobacterium tuberculosis (strain ATCC alkB MRA_3293 A5U7S6 25177/H37Ra) Mycobacterium tuberculosis str. Haarlem TBHG_03188 A4KLE9 Mycobacterium bovis (strain BCG/ alkB BCG_3281c A1KNQ4 Pasteur 1173P2) Mycobacterium bovis 04-303 O216_17560 V2W1E0 Mycobacterium bovis AN5 O217_17270 V2VQT4 Mycobacterium tuberculosis GuangZ0019 alkB GuangZ0019_1145 T5HDB1 Mycobacterium tuberculosis FJ05194 alkB FJ05194_2026 T5H4I2 Mycobacterium tuberculosis ‘98-R604 TBKG_02259 T0EL87 INH-RIF-EM’ Mycobacterium marinum str. Europe MMEU_4939 S7S303 Mycobacterium marinum MB2 MMMB2_4134 S7QZY6 Mycobacterium orygis 112400015 MORY_17288 M8DBT2 Mycobacterium tuberculosis NCGM2209 alkB NCGM2209_3538 G2UTS8 Mycobacterium bovis BCG str. Moreau alkB BCGM_3265c F9UZB9 RDJ Mycobacterium tuberculosis W-148 TBPG_00365 F2VCH4 Mycobacterium tuberculosis CDC1551A TMMG_02400 E9ZP16 Mycobacterium tuberculosis SUMu012 TMLG_02024 E2WM40 Mycobacterium tuberculosis SUMu011 TMKG_02511 E2WA16 Mycobacterium tuberculosis SUMu010 TMJG_03436 E2VYW3 Mycobacterium tuberculosis SUMu009 TMIG_02769 E2VMD7 Mycobacterium tuberculosis SUMu006 TMFG_00461 E2UQS7 Mycobacterium tuberculosis SUMu005 TMEG_03649 E2UEQ4 Mycobacterium tuberculosis SUMu004 TMDG_02087 E2U2V2 Mycobacterium tuberculosis SUMu003 TMCG_01675 E2TRB4 Mycobacterium tuberculosis SUMu002 TMBG_01947 E2TG69 Mycobacterium tuberculosis SUMu001 TMAG_02705 E1HE07 Mycobacterium africanum K85 TBOG_03815 D6FRF3 Mycobacterium tuberculosis CPHL_A TBNG_02887 D6FLF8 Mycobacterium tuberculosis T46 TBLG_03890 D6F9Q1 Mycobacterium tuberculosis T17 TBJG_02010 D5ZLD1 Mycobacterium tuberculosis GM 1503 TBIG_02964 D5Z897 Mycobacterium tuberculosis 02_1987 TBBG_01719 D5YWK4 Mycobacterium tuberculosis EAS054 TBGG_02463 D5YJM0 Mycobacterium tuberculosis T85 TBEG_02389 D5Y8I4 Mycobacterium tuberculosis T92 TBDG_02114 D5XYS2 Mycobacterium tuberculosis C TBCG_03191 A2VP49 Rhodococcus sp. EsD8 EBESD8_35530 N1M6K3 Amycolatopsis orientalis HCCB10007 AORI_4274 R4SU00 Mycobacterium tuberculosis SUMu008 TMHG_02473 E2VD73 Mycobacterium tuberculosis SUMu007 TMGG_02800 E2V1Z1 Mycobacterium tuberculosis 94_M4241A TBAG_02148 D7EUC2 Gordonia amarae NBRC 15530 alkB GOAMR_02_00080 G7GIN7 Rhodococcus rhodochrous ATCC 21198 RR21198_2302 W4A7D8 Amycolatopsis decaplanina DSM 44594 H074_07696 M2XNH0 Mycobacterium sp. 012931 MMSP_4721 S7R3L1 Rhodococcus erythropolis (strain PR4/ alkB RER_07460 C0ZPX6 NBRC 100887) Rhodococcus sp. (strain Q15) alkB1 Q93DN3 Rhodococcus erythropolis CCM2595 O5Y_03630 T1VI31 Rhodococcus sp. P27 N806_28900 U0EPX3 Rhodococcus erythropolis (Arthrobacter alkB1 Q9XAU0 picolinophilus) Rhodococcus qingshengii BKS 20-40 G418_23516 M2V230 Rhodococcus erythropolis SK121 RHOER0001_0742 C3JUT8 Rhodococcus erythropolis DN1 N601_07180 T5HYU5 Nocardia farcinica (strain IFM 10152) NFA_46180 Q5YQS2 Rhodococcus equi NBRC 101255 = C 7 H849_17115 U5DRE7 Shewanella sp. NJ49 alkB1 E3VRS8 Mycobacterium canettii CIPT 140070010 alkB BN42_41302 L0QPN9 Nocardia nova SH22a NONO_c63220 W5TPB1 Rhodococcus equi (strain 103S) alkB REQ_33430 E4WK80 (Corynebacterium equi) Gordonia terrae C-6 GTC6_09699 R7YA99 Nocardioides sp. (strain BAA-499/JS614) Noca_0122 A1SCY2 Gordonia sp. TF6 alkB2 Q5WA49 Hydrocarboniphaga effusa AP103 WQQ_18760 I7ZII6 Gordonia terrae NBRC 100016 alkB GOTRE_037_00320 H5UBE8 Nocardia brasiliensis ATCC 700358 O3I_035145 K0FBU4 Amycolatopsis mediterranei RB B737_6308 T1V895 Amycolatopsis mediterranei (strain S699) AMES_6308 RAM_32810 G0FN68 (Nocardia mediterranei) Amycolatopsis mediterranei (strain U-32) AMED_6400 D8HXC8 Rhodococcus sp. p52 alkB U5S065 Rhodococcus pyridinivorans AK37 AK37_01067 H0JKW2 Rhodococcus pyridinivorans SB3094 Y013_07620 V9XAS5 Janibacter sp. HTCC2649 JNB_17248 A3TPZ2 Gordonia sp. KTR9 KTR9_2914 J9SIP3 Aeromicrobium marinum DSM 15272 HMPREF0063_10220 E2S863 Dietzia cinnamea P4 ES5_02159 E6J5E4 Micromonospora aurantiaca (strain ATCC Micau_3940 D9T1D7 27029/DSM 43813/JCM 10878/NBRC 16125/INA 9442) Dietzia sp. E1 alkB/rub fusion C0LMW6 Rhodococcus ruber BKS 20-38 G352_24171 M2YYB5 Mycobacterium gilvum (strain PYR-GCK) Mflv_3369 A4TAB7 (Mycobacterium flavescens (strain ATCC 700033/PYR-GCK)) Nocardioidaceae bacterium Broad-1 NBCG_03866 E9UYJ8 Rhodococcus rhodochrous ATCC 21198 RR21198_2485 W4A610 Salinisphaera shabanensis E1L3A SSPSH_001855 U2E637 Rhodococcus erythropolis (strain PR4/ alkB RER_54580 C0ZSH4 NBRC 100887) Corynebacterium falsenii DSM 44353 CFAL_02965 W5WPK1 Rhodococcus erythropolis CCM2595 O5Y_25995 T1VVR3 gamma proteobacterium BDW918 DOK_04793 I2JMI2 Rhodococcus sp. P27 N806_02390 U0DZR9 Rhodococcus erythropolis DN1 N601_00885 T5IAL6 Rhodococcus erythropolis SK121 RHOER0001_2104 C3JNE0 Rhodococcus qingshengii BKS 20-40 G418_13569 M2WBK9

In some embodiments, the disclosure provides methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is a long-chain alkane hydroxylase. In some embodiments, the long-chain alkane hydroxylase is selected from Table 5 or a variant thereof having at least 90% identity thereto.

TABLE 5 Long chain alkane hydroxylase enzymes capable of catalyzing selective terminal alkene hydroxylation. Species Origin Gene names Accession No Geobacillus thermodenitrificans (strain NG80-2) ladA GTNG_3499 A4IU28 Geobacillus stearothermophilus (Bacillus A8DC15 stearothermophilus) Paenibacillus sp. JCM 10914 JCM10914_4324 V9GEW8 Bacillus methanolicus MGA3 MGA3_06970 I3E8X7 Geobacillus sp. (strain Y4.1MC1) GY4MC1_0235 E3IA76 Geobacillus thermoglucosidans TNO-09.020 GT20_0226 I0U377 Geobacillus thermoglucosidasius (strain C56-YS93) Geoth_0249 F8CWH6 Bacillus methanolicus PB1 PB1_11994 I3DVL0 Alicyclobacillus acidoterrestris ATCC 49025 N007_16655 T0BMR6 Bhargavaea cecembensis DSE10 ntaA_1 C772_00943 M7P9Z6 Bacillus sp. 1NLA3E B1NLA3E_02955 N0AV94 Burkholderia graminis C4D1M BgramDRAFT_6080 B1G9N8 Burkholderia thailandensis H0587 BTL_4503 W6BDT9 Planomicrobium glaciei CHR43 G159_18855 W3A818 Burkholderia thailandensis E444 BTJ_3656 W6C8D0 Burkholderia thailandensis 2002721723 BTQ_5029 W6BNA4 Burkholderia pseudomallei (strain K96243) BPSS0686 Q63MH2 Burkholderia mallei (strain ATCC 23344) BMAA1146 Q62BX8 Burkholderia thailandensis (strain E264/ATCC 700388/ BTH_II1741 Q2T4G4 DSM 13276/CIP 106301) Burkholderia pseudomallei BPC006 BPC006_II0968 K7QBE8 Burkholderia pseudomallei 1106b BURPS1106B_1056 C5ZS78 Burkholderia pseudomallei MSHR346 GBP346_B0209 C4I1H5 Burkholderia pseudomallei (strain 1106a) BURPS1106A_A0931 A3P3Q7 Burkholderia mallei (strain NCTC 10247) BMA10247_A1520 A3MEL8 Burkholderia mallei (strain NCTC 10229) BMA10229_0093 A2RW50 Burkholderia pseudomallei MSHR338 M218_32405 W1LX20 Burkholderia mallei PRL-20 BMAPRL20_0872 C5N9G7 Burkholderia mallei GB8 horse 4 BMAGB8_A1284 C4B2F1 Burkholderia pseudomallei Pakistan 9 BUH_5241 C0Y1N2 Burkholderia pseudomallei 576 BUC_5105 B7CGH3 Burkholderia pseudomallei S13 BURPSS13_T0065 B1H503 Burkholderia mallei ATCC 10399 BMA10399_L0048 A9LC22 Burkholderia pseudomallei Pasteur 52237 BURPSPAST_J0304 A8KQQ8 Burkholderia pseudomallei 406e BURPS406E_G0092 A8EKE5 Burkholderia mallei JHU BMAJHU_I0303 A5XK99 Burkholderia mallei 2002721280 BMA721280_L0585 A5TFQ0 Alicyclobacillus acidoterrestris ATCC 49025 N007_09450 T0BMI0 Burkholderia pseudomallei MSHR305 BDL_3916 S5NPF6 Burkholderia pseudomallei MSHR146 BBN_4086 W0Q1C4 Burkholderia pseudomallei MSHR511 BBQ_5508 W0MJC7 Burkholderia pseudomallei NAU20B-16 BBS_5466 V9YUR9 Burkholderia pseudomallei NCTC 13178 BBJ_4354 V9YFT0 Burkholderia pseudomallei NCTC 13179 BBK_3804 U5V4E4 Burkholderia pseudomallei MSHR1043 D512_19607 M7EBY4 Burkholderia pseudomallei 1655 BURPS1655_I0183 B2H6F2 Burkholderia pseudomallei 305 BURPS305_5546 A4LI20 Segniliparus rugosus ATCC BAA-974 HMPREF9336_02889 E5XTR7 Burkholderia pseudomallei 1026b BP1026B_II0759 I1WRX2 Burkholderia pseudomallei 354a BP354A_4019 I2MG65 Burkholderia pseudomallei 354e BP354E_3240 I2M2Q7 Burkholderia pseudomallei 1026a BP1026A_2436 I2L127 Burkholderia pseudomallei 1258b BP1258B_3899 I2KY00 Burkholderia pseudomallei 1258a BP1258A_3523 I2KWT0 Pseudomonas putida (strain DOT-T1E) T1E_2746 I7B0Q5 Pseudomonas putida ND6 YSA_09788 I3V2W3 Pseudomonas putida TRO1 C206_18269 N9VYA0 Pseudomonas putida LS46 PPUTLS46_018911 M7RI48 Burkholderia graminis C4D1M BgramDRAFT_6182 B1G9Y6 Burkholderia phytofirmans (strain DSM 17436/PsJN) Bphyt_4538 B2TDZ4 Bhargavaea cecembensis DSE10 moxC_3 C772_02411 M7NEH3 Burkholderia thailandensis MSMB121 BTI_5494 N0APC1 Burkholderia pseudomallei (strain 668) BURPS668_A1016 A3NI44 Burkholderia pseudomallei (strain 1710b) BURPS1710b_A2257 Q3JG95 Burkholderia pseudomallei 1710a BURPS1710A_A0072 C6U1I8 Planomicrobium glaciei CHR43 G159_14295 W3AA87 Burkholderia thailandensis MSMB43 A33K_16732 I6AD68 Pseudomonas sp. GM50 PMI30_04278 J3GFD6 Pseudomonas fluorescens BBc6R8 MHB_001910 V7EA47 Pseudomonas sp. Ag1 A462_06954 J0YEG1 Pseudomonas sp. GM102 PMI18_00569 J2VSE5 Pseudomonas fluorescens (strain SBW25) PFLU_3858 C3JYC1 Pseudomonas sp. (strain M1) PM1_0212365 W5IVB1 Pseudomonas sp. TKP U771_20325 V9R055 Pseudomonas putida (strain F1/ATCC 700007) Pput_3007 A5W4S5 Pseudomonas putida (strain GB-1) PputGB1_1120 B0KS73 Azotobacter vinelandii CA6 seuA AvCA6_43810 M9YDA5 Azotobacter vinelandii CA seuA AvCA_43810 M9Y6B1 Azotobacter vinelandii (strain DJ/ATCC BAA-1303) seuA Avin_43810 C1DGK6 Pseudomonas brassicacearum (strain NFM421) PSEBR_a2282 F2KFH4 Pseudomonas fluorescens Q8r1-96 PflQ8_2313 I4KKG5 Klebsiella oxytoca E718 A225_4709 I6X485 Pseudomonas putida (strain KT2440) PP_2746 Q88JA3 Pseudomonas fluorescens BBc6R8 MHB_002244 V7E7E4 Pseudomonas fluorescens Q2-87 PflQ2_2259 J2EFB8 Pseudomonas sp. Ag1 A462_04671 J0PSS9 Klebsiella oxytoca MGH 42 L388_04093 V3KYZ2 Klebsiella oxytoca 10-5245 HMPREF9689_03721 H3M9T3 Klebsiella oxytoca 10-5243 HMPREF9687_03258 H3LSS6 Klebsiella oxytoca (strain ATCC 8724/DSM 4798/ KOX_01240 G8WD25 JCM 20051/NBRC 3318/NRRL B-199/KCTC 1686) Streptomyces himastatinicus ATCC 53653 SSOG_01846 D9WSJ3 Klebsiella oxytoca MGH 28 L374_04760 V3PRZ9 Klebsiella oxytoca 10-5250 HMPREF9694_02187 H3N1Z4 Klebsiella sp. OBRC7 HMPREF1144_4230 J8VYP0 Klebsiella oxytoca 10-5242 HMPREF9686_03185 H3LCA0 Pantoea ananatis LMG 5342 soxA PANA5342_1855 G9ARF4 Pantoea ananatis PA13 PAGR_g1792 G7UD55 Pantoea ananatis (strain AJ13355) soxA PAJ_1557 F2EW92 Pantoea ananatis (strain LMG 20103) soxA PANA_2246 D4GGW6 Pantoea ananatis BRT175 L585_00145 U4W7P0 Segniliparus rotundus (strain ATCC BAA-972/CDC Srot_2598 D6ZC64 1076/CIP 108378/DSM 44985/JCM 13578) Pantoea stewartii subsp. stewartii DC283 CKS_1871 H3RFH9 Pantoea stewartii subsp. stewartii DC283 CKS_1871 H3RFH9 Rhodococcus opacus M213 WSS_A14179 K8XV73 Klebsiella pneumoniae DMC0799 H217_2899 S7AJY1 Klebsiella pneumoniae 700603 KP700603_18582 M7P910 Klebsiella sp. MS 92-3 HMPREF9538_02211 F3Q553 Klebsiella pneumoniae CG43 D364_16040 U5MF64 Klebsiella pneumoniae subsp. pneumoniae 1084 A79E_0950 K4HBM3 Klebsiella pneumoniae subsp. pneumoniae (strain KPHS_42240 G8VT84 HS11286) Klebsiella pneumoniae KCTC 2242 KPN2242_18760 G0GTG2 Klebsiella pneumoniae NB60 X657_3893 W7K535 Klebsiella pneumoniae EGD-HP19-C N035_09715 W1LTN4 Escherichia coli ISC56 W1HC22 Klebsiella pneumoniae IS33 W1CX87 Klebsiella pneumoniae subsp. pneumoniae BJ1-GA KPST380_90022 W0YH64 Klebsiella pneumoniae subsp. pneumoniae SA1 KPST86_100232 W0XPM0 Klebsiella pneumoniae subsp. pneumoniae T69 SB4536_310004 W0XIP7 Klebsiella pneumoniae MGH 18 L364_01157 V3UHS5 Klebsiella pneumoniae MGH 17 L363_03338 V3SPP7 Klebsiella pneumoniae MGH 21 L367_03372 V3RJW0 Klebsiella pneumoniae MGH 19 L365_03262 V3RDD9 Klebsiella pneumoniae MGH 32 L378_01018 V3P5E6 Klebsiella pneumoniae MGH 30 L376_01094 V3NNG8 Klebsiella pneumoniae MGH 40 L386_03550 V3MB44 Klebsiella pneumoniae MGH 36 L382_03249 V3M7N6 Klebsiella pneumoniae BWH 28 L399_01071 V3JYS4 Klebsiella pneumoniae BWH 30 L401_03358 V3IHX3 Klebsiella pneumoniae UCICRE 2 L413_01241 V3H9M1 Klebsiella pneumoniae UCICRE 7 L418_00976 V3FW02 Klebsiella pneumoniae UCICRE 6 L417_03180 V3FI89 Klebsiella pneumoniae BIDMC 21 L457_03247 V3DWM2 Klebsiella pneumoniae BIDMC 22 L458_03227 V3DGZ8 Klebsiella pneumoniae BIDMC 24 L460_03188 V3BDU6 Klebsiella pneumoniae BIDMC 25 L461_03214 V3B499 Klebsiella pneumoniae BIDMC 40 L477_03188 V3A962 Klebsiella pneumoniae BIDMC 36 L473_03258 V3A6I8 Klebsiella pneumoniae BIDMC 41 L478_00374 V2Z7W2 Klebsiella pneumoniae BIDMC 12C L441_03468 U7BFN1 Klebsiella pneumoniae BIDMC 18C L450_03424 U7AVL5 Klebsiella pneumoniae BIDMC 16 L445_03710 U7AGB1 Enterococcus gallinarum EGD-AAK12 N036_14515 U1CX13 Klebsiella pneumoniae subsp. pneumoniae MP14 KKPNMP14_39700 S8A752 Klebsiella pneumoniae subsp. pneumoniae UKKV901664_39580 S7YC36 UKKV901664 Klebsiella pneumoniae 120_1020 J048_0227 S7I734 Klebsiella pneumoniae 140_1040 J046_0551 S7HZ61 Klebsiella pneumoniae 280_1220 J049_0615 S7H6G5 Klebsiella pneumoniae 160_1080 J047_06104 S7FI27 Klebsiella pneumoniae UHKPC06 H228_0695 S7F6A3 Klebsiella pneumoniae UHKPC67 H212_0084 S7EIH5 Klebsiella pneumoniae UHKPC02 H229_0083 S7EFH7 Klebsiella pneumoniae UHKPC17 H225_0083 S7E3F9 Klebsiella pneumoniae UHKPC31 H227_0223 S7E0F6 Klebsiella pneumoniae UHKPC59 H223_2084 S7DJY5 Klebsiella pneumoniae UHKPC18 H226_0627 S7CZN2 Klebsiella pneumoniae UHKPC61 H220_0228 S7CKP4 Klebsiella pneumoniae UHKPC07 H224_0554 S7C1T8 Klebsiella pneumoniae DMC1316 H219_1515 S7C0U0 Klebsiella pneumoniae UHKPC33 H222_0227 S7BH54 Klebsiella pneumoniae DMC1097 H218_2245 S7A1J0 Klebsiella pneumoniae UHKPC96 H215_0710 S6YYA8 Klebsiella pneumoniae UHKPC77 H214_0083 S6YU31 Klebsiella pneumoniae UHKPC28 H209_0679 S6YQS7 Klebsiella pneumoniae UHKPC69 H213_0083 S6YBZ0 Klebsiella pneumoniae UHKPC47 H211_0128 S6XBP3 Klebsiella pneumoniae UHKPC32 H242_0078 S2J6Y7 Klebsiella pneumoniae UHKPC48 H221_0076 S2I2J3 Klebsiella pneumoniae DMC0526 H216_2445 S2I0S2 Klebsiella pneumoniae VAKPC278 H247_0907 S2H7F7 Klebsiella pneumoniae UHKPC29 H241_0227 S2GQ63 Klebsiella pneumoniae UHKPC05 H210_0554 S2G118 Klebsiella pneumoniae UHKPC45 H239_0077 S2FVN7 Klebsiella pneumoniae UHKPC 52 H234_0218 S2FQ55 Klebsiella pneumoniae 646_1568 J054_0227 S2E5R5 Klebsiella pneumoniae 540_1460 J053_0083 S2E2M9 Klebsiella pneumoniae 440_1540 J051_2140 S2CWI6 Klebsiella pneumoniae 500_1420 J052_0542 S2CKG8 Klebsiella pneumoniae VAKPC309 H252_1202 S2C6A5 Klebsiella pneumoniae KP-11 H254_0775 S2BTB1 Klebsiella pneumoniae 361_1301 J050_2658 S2B565 Klebsiella pneumoniae VAKPC297 H251_0083 S2ACA5 Klebsiella pneumoniae VAKPC270 H249_0897 S1ZBB5 Klebsiella pneumoniae VAKPC280 H248_0984 S1Z9L1 Klebsiella pneumoniae VAKPC276 H250_1158 S1Z4C6 Klebsiella pneumoniae VAKPC269 H246_1198 S1YJN2 Klebsiella pneumoniae VAKPC254 H245_0083 S1XZP2 Klebsiella pneumoniae UHKPC22 H240_0083 S1XYX9 Klebsiella pneumoniae UHKPC04 H243_0549 S1X5H6 Klebsiella pneumoniae VAKPC252 H244_3523 S1WWW4 Klebsiella pneumoniae UHKPC26 H236_0227 S1W5H8 Klebsiella pneumoniae UHKPC27 H233_0552 S1VUY3 Klebsiella pneumoniae UHKPC24 H235_0228 S1V9Y4 Klebsiella pneumoniae UHKPC01 H231_1154 S1V1B9 Klebsiella pneumoniae UHKPC81 H232_2378 S1TWU9 Klebsiella pneumoniae UHKPC40 H207_0083 S1TR15 Klebsiella pneumoniae UHKPC09 H230_0227 S1TQU1 Klebsiella pneumoniae KP-7 H253_1042 S1T453 Klebsiella pneumoniae UHKPC23 H208_0755 R9BIA6 Klebsiella pneumoniae subsp. pneumoniae KpMDU1 C210_21528 N9SXP2 Klebsiella pneumoniae ATCC BAA-1705 KPBAA1705_02256 M7QWX8 Klebsiella pneumoniae ATCC BAA-2146 G000_17665 Kpn2146_4394 M7PZV3 Klebsiella pneumoniae VA360 MTE2 213 M5T2W9 Klebsiella pneumoniae RYC492 KPRYC492_05065 M5Q5H7 Klebsiella pneumoniae RYC492 KPRYC492_05065 M5Q5H7 Klebsiella pneumoniae subsp. pneumoniae KpQ3 B819_29014 M5GIZ6 Klebsiella pneumoniae subsp. pneumoniae Ecl8 BN373_37921 K4UK89 Klebsiella pneumoniae subsp. pneumoniae WGLW5 HMPREF1308_03340 K1NXD5 Klebsiella pneumoniae subsp. pneumoniae WGLW3 HMPREF1307_01233 K1NCK1 Klebsiella pneumoniae subsp. pneumoniae WGLW1 HMPREF1305_01058 K1MMN7 Klebsiella pneumoniae subsp. pneumoniae KPNIH23 KPNIH23_01714 J2W4N5 Klebsiella pneumoniae subsp. pneumoniae KPNIH21 KPNIH21_18909 J2UUP0 Klebsiella pneumoniae subsp. pneumoniae KPNIH18 KPNIH18_04648 J2TP42 Klebsiella pneumoniae subsp. pneumoniae KPNIH17 KPNIH17_07852 J2SZ94 Klebsiella pneumoniae subsp. pneumoniae KPNIH9 KPNIH9_07912 J2PY88 Klebsiella pneumoniae subsp. pneumoniae KPNIH6 KPNIH6_12977 J2NIU0 Klebsiella pneumoniae subsp. pneumoniae KPNIH1 KPNIH1_04615 J2MHH3 Klebsiella pneumoniae subsp. pneumoniae KPNIH22 KPNIH22_01396 J2KA06 Klebsiella pneumoniae subsp. pneumoniae KPNIH19 KPNIH19_02887 J2JA47 Klebsiella pneumoniae subsp. pneumoniae KPNIH16 KPNIH16_07898 J2HIQ1 Klebsiella pneumoniae subsp. pneumoniae KPNIH14 KPNIH14_01932 J2GTK1 Klebsiella pneumoniae subsp. pneumoniae KPNIH11 KPNIH11_05794 J2G1J7 Klebsiella pneumoniae subsp. pneumoniae KPNIH2 KPNIH2_14379 J2BUC4 Klebsiella pneumoniae subsp. pneumoniae KPNIH20 KPNIH20_08348 J2BFJ4 Klebsiella pneumoniae subsp. pneumoniae KPNIH12 KPNIH12_01874 J1YXJ0 Klebsiella pneumoniae subsp. pneumoniae KPNIH10 KPNIH10_07382 J1X9E8 Klebsiella pneumoniae subsp. pneumoniae KPNIH8 KPNIH8_09376 J1WTX7 Klebsiella pneumoniae subsp. pneumoniae KPNIH7 KPNIH7_03054 J1WDZ3 Klebsiella pneumoniae subsp. pneumoniae KPNIH5 KPNIH5_11286 J1V7M9 Klebsiella pneumoniae subsp. pneumoniae KPNIH4 KPNIH4_01334 J1UFY7 Klebsiella sp. 4_1_44FAA HMPREF1024_02306 G9REB7 Klebsiella pneumoniae JM45 N559_1083 S5YDY6 Klebsiella pneumoniae subsp. pneumoniae Kp13 KP13_02362 V9ZFM9 Klebsiella pneumoniae subsp. rhinoscleromatis ATCC HMPREF0484_1763 C8T2C2 13884 Klebsiella pneumoniae subsp. pneumoniae ST258-K26BO BN426_1797 K4RX40 Klebsiella variicola (strain At-22) Kvar_0908 D3RIP8 Klebsiella pneumoniae (strain 342) KPK_0975 B5XUZ5 Klebsiella pneumoniae MGH 20 L366_04030 V3R3V0 Klebsiella pneumoniae UCICRE 10 L421_04096 V3DSZ3 Klebsiella sp. KTE92 A1WC_04002 R8X357 Klebsiella pneumoniae hvKP1 G057_03698 M2A8M6 Mycobacterium hassiacum DSM 44199 C731_0966 K5B980 Klebsiella pneumoniae MGH 48 L394_03318 V3J564 Pantoea vagans (strain C9-1) (Pantoea agglomerans Pvag_pPag10056 E1PKF9 (strain C9-1)) Klebsiella pneumoniae IS22 W1BJB8 Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044 KP1_4424 C4XCS7 Burkholderia sp. CCGE1001 BC1001_4137 E8YTA8 Microvirga lotononidis MicloDRAFT_00046760 I4YVV6 Burkholderia phenoliruptrix BR3459a BUPH_00719 K0DVZ1 Pseudomonas cichorii JBC1 PCH70_03420 W0H3V5 Burkholderia sp. (strain CCGE1003) BC1003_5279 E1TDZ6 Pseudomonas protegens CHA0 soxA1 PFLCHA0_c02440 R4QZ42 Herbaspirillum sp. CF444 PMI16_04881 J2L7C4 Pseudomonas fluorescens (strain Pf-5/ATCC BAA-477) PFL_0243 Q4KK44 Bacillus megaterium WSH-002 BMWSH_4371 G2RTT4 Pseudomonas sp. GM30 PMI25_001642 W6W1D9 Pseudomonas sp. GM78 PMI35_05139 J3D9L9 Pseudomonas sp. GM60 PMI32_02771 J2U6I1 Pseudomonas sp. FH1 H096_21398 W2DLN3 Pseudomonas sp. GM41(2012) PMI27_000125 W6VAV2 Pseudomonas sp. GM67 PMI33_04861 J2TPB1 Pseudomonas fluorescens EGD-AQ6 O204_08695 U1U9U7 Pseudomonas sp. CF161 CF161_31485 S6JVW1 Pseudomonas fluorescens BRIP34879 A986_05371 L7HKJ7 Pseudomonas sp. Lz4W B195_18896 M5QDB7 Collimonas fungivorans (strain Ter331) CFU_2748 G0A9F2 Pseudomonas poae RE*1-1-14 H045_11420 M4K052 Pseudomonas fluorescens BBc6R8 MHB_005864 V7DXC0 Pseudomonas sp. Lz4W B195_14957 M5QFC1 Pseudomonas sp. GM24 PMI23_03232 J2QII9 Pseudomonas sp. GM16 PMI19_05169 J2MFI6 Rhizobium sp. CF080 PMI07_000401 W6W3M6 Pseudomonas sp. FH1 H096_13584 W2DVJ0 Pseudomonas sp. GM25 PMI24_00141 J2Q9Q8 Rhizobium leguminosarum bv. trifolii (strain WSM2304) Rleg2_6510 B6A4D5 Pseudomonas sp. G5(2012) PG5_63250 S2FDS8 Pseudomonas chlororaphis O6 PchlO6_2640 I4XU61 Pseudomonas protegens CHA0 soxA3 PFLCHA0_c26840 R4R5M1 Pseudomonas fluorescens (strain Pf-5/ATCC BAA-477) PFL_2617 Q4KDF9 Rhizobium leguminosarum bv. trifolii WSM597 Rleg9DRAFT_0832 I9N5T8 Bacillus megaterium (strain DSM 319) BMD_1582 D5DC52 Pseudomonas fluorescens WH6 PFWH6_3643 E2XUD7 Rhizobium sp. Pop5 RCCGEPOP_16608 K0VYN0 Bacillus megaterium (strain ATCC 12872/QMB1551) BMQ_0870 D5E197 Pseudomonas cichorii JBC1 PCH70_26220 W0HAE8 Pseudomonas sp. TKP U771_03925 V9QRK7 Pseudomonas aeruginosa C41 Q088_02376 U8DE40 Pseudomonas aeruginosa 62 P997_00130 U9DU73 Pseudomonas aeruginosa BL19 Q073_02117 U8H8T3 Pseudomonas aeruginosa YL84 AI22_19865 W5VAE7 Pseudomonas aeruginosa SCV20265 SCV20265_2995 V9U1K6 Pseudomonas aeruginosa LES431 T223_15220 V9T819 Pseudomonas aeruginosa MTB-1 U769_13585 V5SWN7 Pseudomonas aeruginosa PA1R PA1R_gp0125 U6AQA7 Pseudomonas aeruginosa PA1 PA1S_gp0125 U6A6M8 Pseudomonas aeruginosa PAO1-VE13 N297_2400 U5RPB5 Pseudomonas aeruginosa PAO1-VE2 N296_2400 U5R2L8 Pseudomonas aeruginosa c7447m M802_2397 T2EJL9 Pseudomonas aeruginosa RP73 M062_12135 R9ZF43 Pseudomonas aeruginosa (strain ATCC 15692/PAO1/ PA2326 Q9I1F2 1C/PRS 101/LMG 12228) Pseudomonas aeruginosa (strain UCBPP-PA14) PA14_34540 Q02MC3 Pseudomonas aeruginosa B136-33 G655_13420 M9S636 Pseudomonas aeruginosa DK2 PADK2_13640 I6SJ32 Pseudomonas aeruginosa (strain LESB58) PLES_29781 B7V8Z7 Pseudomonas aeruginosa (strain PA7) PSPA7_2933 A6V5F8 Pseudomonas aeruginosa (strain PA7) PSPA7_2933 A6V5F8 Pseudomonas aeruginosa DHS29 V441_13990 W1QXR3 Pseudomonas aeruginosa MH38 P38_3412 W0WGT3 Pseudomonas aeruginosa VRFPA06 V527_13850 V8HJN2 Pseudomonas aeruginosa VRFPA08 X922_29130 V8DQV8 Pseudomonas aeruginosa DHS01 DPADHS01_13190 V4WR77 Pseudomonas aeruginosa VRFPA01 G039_0203575 V4QMQ4 Pseudomonas aeruginosa HB15 PA15_0330520 V4MN40 Pseudomonas aeruginosa M8A.3 Q082_00075 U9SHI5 Pseudomonas aeruginosa CF27 Q003_00104 U9RU06 Pseudomonas aeruginosa MSH10 Q000_02112 U9RT23 Pseudomonas aeruginosa CF127 Q001_02232 U9RQB8 Pseudomonas aeruginosa CF5 Q004_02036 U9R042 Pseudomonas aeruginosa S54485 Q007_00776 U9QQE4 Pseudomonas aeruginosa BWHPSA007 Q020_00157 U9PK67 Pseudomonas aeruginosa BWHPSA009 Q022_02698 U9NGB4 Pseudomonas aeruginosa BWHPSA008 Q021_00149 U9NF67 Pseudomonas aeruginosa BWHPSA010 Q023_01638 U9MXZ6 Pseudomonas aeruginosa BWHPSA015 Q028_00447 U9MBW2 Pseudomonas aeruginosa BWHPSA016 Q029_01714 U9LQK4 Pseudomonas aeruginosa BL03 Q057_00105 U9LB58 Pseudomonas aeruginosa BL01 Q055_02736 U9KLQ0 Pseudomonas aeruginosa BL02 Q056_06394 U9JUP8 Pseudomonas aeruginosa BL05 Q059_02100 U9JF28 Pseudomonas aeruginosa BL06 Q060_06378 U9IJ92 Pseudomonas aeruginosa BL21 Q075_03038 U9GQQ1 Pseudomonas aeruginosa BL23 Q077_03073 U9FQH5 Pseudomonas aeruginosa BL24 Q078_06288 U9EQY5 Pseudomonas aeruginosa M8A.4 Q083_01720 U9ECA2 Pseudomonas aeruginosa MSH3 P999_02290 U9D2B6 Pseudomonas aeruginosa X24509 Q005_02076 U9CCX5 Pseudomonas aeruginosa UDL Q006_01725 U9C927 Pseudomonas aeruginosa CF18 Q002_02068 U9BVH8 Pseudomonas aeruginosa 19660 Q010_02159 U9AF43 Pseudomonas aeruginosa X13273 Q013_02044 U8Z334 Pseudomonas aeruginosa S35004 Q012_06204 U8YF61 Pseudomonas aeruginosa BWHPSA001 Q014_02765 U8YAB2 Pseudomonas aeruginosa BWHPSA003 Q016_02194 U8XR83 Pseudomonas aeruginosa BWHPSA002 Q015_02292 U8XP62 Pseudomonas aeruginosa BWHPSA004 Q017_02030 U8X7A0 Pseudomonas aeruginosa BWHPSA005 Q018_03069 U8W6E8 Pseudomonas aeruginosa BWHPSA011 Q024_01957 U8VA48 Pseudomonas aeruginosa BWHPSA013 Q026_03028 U8URW4 Pseudomonas aeruginosa BWHPSA012 Q025_02769 U8UQP2 Pseudomonas aeruginosa BWHPSA014 Q027_01719 U8TK96 Pseudomonas aeruginosa BWHPSA017 Q030_05589 U8SKH8 Pseudomonas aeruginosa BWHPSA020 Q033_02593 U8S609 Pseudomonas aeruginosa BWHPSA019 Q032_03133 U8RPR9 Pseudomonas aeruginosa BWHPSA022 Q035_01895 U8R8U4 Pseudomonas aeruginosa BWHPSA023 Q036_00320 U8R6B4 Pseudomonas aeruginosa BWHPSA021 Q034_02035 U8R1N4 Pseudomonas aeruginosa BWHPSA025 Q038_01757 U8PR31 Pseudomonas aeruginosa BWHPSA024 Q037_02761 U8PP93 Pseudomonas aeruginosa BWHPSA027 Q040_02049 U8N8N1 Pseudomonas aeruginosa BL07 Q061_01439 U8LYS6 Pseudomonas aeruginosa BL04 Q058_06192 U8LL05 Pseudomonas aeruginosa BL11 Q065_03099 U8K8S5 Pseudomonas aeruginosa BL10 Q064_02801 U8JQ84 Pseudomonas aeruginosa BL15 Q069_01997 U8IMR3 Pseudomonas aeruginosa BL16 Q070_01957 U8IID0 Pseudomonas aeruginosa BL18 Q072_02105 U8H8J8 Pseudomonas aeruginosa M8A.2 Q081_01961 U8FTG3 Pseudomonas aeruginosa M8A.1 Q080_04721 U8FHJ8 Pseudomonas aeruginosa M9A.1 Q084_05530 U8EPH5 Pseudomonas aeruginosa C20 Q085_03119 U8EML6 Pseudomonas aeruginosa C23 Q086_03122 U8EJ68 Pseudomonas aeruginosa C40 Q087_02201 U8DKJ1 Pseudomonas aeruginosa C48 Q089_02700 U8CPW7 Pseudomonas aeruginosa C51 Q090_05806 U8BVH7 Pseudomonas aeruginosa CF77 Q092_01904 U8BA80 Pseudomonas aeruginosa C52 Q091_05688 U8AZD2 Pseudomonas aeruginosa CF614 Q093_06204 U8ACM4 Pseudomonas aeruginosa VRFPA04 P797_30195 U5AHY5 Pseudomonas aeruginosa HB13 PA13_1029315 U1E3A4 Pseudomonas aeruginosa MSH-10 L346_02111 S0IJJ1 Pseudomonas aeruginosa PA14 CIA_02266 S0I9C6 Pseudomonas aeruginosa PAK PAK_02986 S0I695 Pseudomonas sp. P179 HMPREF1224_05539 N2DDM6 Pseudomonas aeruginosa str. Stone 130 HMPREF1223_07114 N2D7D2 Pseudomonas aeruginosa PA21_ST175 H123_24636 M3AW72 Pseudomonas aeruginosa E2 P998_02032 PAE2_2544 K1DHT6 Pseudomonas aeruginosa ATCC 25324 PABE173_3188 K1DD82 Pseudomonas aeruginosa CI27 PACI27_2786 K1CTB3 Pseudomonas aeruginosa ATCC 700888 PABE177_2660 K1CGR7 Pseudomonas aeruginosa ATCC 14886 PABE171_3115 K1BXJ5 Pseudomonas aeruginosa PADK2_CF510 CF510_22344 I1ACS3 Pseudomonas aeruginosa MPAO1/P2 O1Q_15090 H3TFC3 Pseudomonas aeruginosa MPAO1/P1 O1O_28545 H3T6G4 Pseudomonas sp. 2_1_26 HMPREF1030_05556 G5G1F3 Pseudomonas aeruginosa 2192 PA2G_01431 A3LB74 Pseudomonas aeruginosa C3719 PACG_01235 A3KU95 Erwinia billingiae (strain Eb661) EbC_20720 D8MRZ6 Xanthomonas axonopodis pv. citri (strain 306) XAC0855 Q8PP33 Xanthomonas citri subsp. citri Aw12879 XCAW_03724 M4W2T5 Xanthomonas axonopodis Xac29-1 XAC29_04355 M4U7K3 Xanthomonas citri pv. mangiferaeindicae LMG 941 ladA XMIN_2789 H8FHG1 Xanthomonas axonopodis pv. punicae str. LMG 859 ladA XAPC_728 H1XCV7 Leifsonia aquatica ATCC 14665 N136_01626 U2TBF7 Serratia marcescens subsp. marcescens Db11 SMDB11_2421 V6A0D9 Pseudomonas aeruginosa VRFPA05 T266_33830 V4WJP9 Pseudomonas aeruginosa BL22 Q076_01761 U9GCW5 Pseudomonas aeruginosa BL22 Q076_01761 U9GCW5 Xanthomonas axonopodis pv. malvacearum str. MOU_00060 K8GBN4 GSPB1386 Pseudomonas aeruginosa VRFPA07 X778_28580 V8E3G0 Pseudomonas aeruginosa BL20 Q074_02826 U9HSV9 Pseudomonas aeruginosa BL25 Q079_01143 U9F0W8 Pseudomonas aeruginosa BL09 Q063_00187 U8L2Y0 Serratia marcescens WW4 SMWW4_v1c31920 L7ZQQ5 Serratia marcescens VGH107 F518_24469 M3BTM0 Pseudomonas aeruginosa BWHPSA018 Q031_00379 U8TSK3 Pseudomonas aeruginosa M18 PAM18_2715 G2L1H6 Pseudomonas aeruginosa BL12 Q066_03852 U9I855 Pseudomonas aeruginosa BWHPSA028 Q041_02218 U8NES6 Pseudomonas aeruginosa WC55 L683_26830 T5KSU5 Pseudomonas aeruginosa NCMG1179 NCGM1179_2739 G2U5R3 Rhodococcus erythropolis SK121 RHOER0001_2299 C3JDL9 Pseudomonas aeruginosa VRFPA03 M770_16185 W1MK34 Pseudomonas aeruginosa BL13 Q067_03184 U9I925 Serratia marcescens EGD-HP20 N040_11055 U1TLQ0 Pseudomonas aeruginosa NCGM2.S1 NCGM2_3338 G4LI50 Pseudomonas aeruginosa 39016 PA39016_002700003 E3A2U8 Pseudomonas aeruginosa MH27 PAMH27_2887 V6AFD9 Pseudomonas aeruginosa JJ692 Q008_02805 U9PMT7 Pseudomonas aeruginosa 6077 Q011_02150 U9ATK4 Pseudomonas aeruginosa U2504 Q009_02593 U9AAM5 Pseudomonas aeruginosa BWHPSA006 Q019_02936 U8VL16 Pseudomonas aeruginosa BL08 Q062_04340 U8KSZ8 Pseudomonas aeruginosa BL14 Q068_02182 U8JUF2 Pseudomonas aeruginosa BL17 Q071_02971 U8H8J5 Pseudomonas aeruginosa PA45 H734_07342 N4W202 Rhodococcus erythropolis CCM2595 O5Y_21155 T1VSG7 Rhodococcus sp. P27 N806_09240 U0ED84 Kosakonia radicincitans DSM 16656 Y71_0158 J1QW00 Rhodococcus erythropolis (strain PR4/NBRC 100887) RER_45000 C0ZMF0 Klebsiella pneumoniae MGH 46 L392_03264 V3LZ98 Klebsiella pneumoniae MGH 44 L390_02205 V3JUR2 Klebsiella pneumoniae UCICRE 4 L415_03363 V3FXF6 Klebsiella pneumoniae 303K N598_24365 U6T101 Klebsiella pneumoniae UHKPC179 H238_2267 S7F9A7 Klebsiella pneumoniae UHKPC57 H237_2247 S2EDB5 Klebsiella pneumoniae JHCK1 MTE1_213 M3U9Q5 Klebsiella pneumoniae subsp. pneumoniae WGLW2 HMPREF1306_03733 K1NBI6 Klebsiella pneumoniae UCICRE 14 L425_03054 V3CJD9 Rhodococcus qingshengii BKS 20-40 G418_04858 M2XMT9 Pantoea sp. Sc1 S7A_19914 H8DUB8 Klebsiella sp. 1_1_55 HMPREF0485_02899 D6GIG4 Pantoea agglomerans Tx10 L584_13665 U4VW62 Escherichia coli 909957 HMPREF1619_02817 V0B421 Klebsiella pneumoniae KP-1 KLP1_1662 U2ABR1 Rhodococcus erythropolis DN1 N601_05680 T5I9L8 Klebsiella pneumoniae UCICRE 8 L419_03300 V3F3T1 Brenneria sp. EniD312 BrE312_1717 G7LVX2 Klebsiella pneumoniae BIDMC 23 L459_03205 V3BAE8 Raoultella ornithinolytica B6 RORB6_23555 M9W8P0 Klebsiella oxytoca 10-5246 HMPREF9690_03902 H3MRJ7 Pantoea agglomerans 299R F385_1445 L7BV82 Pantoea sp. aB PanABDRAFT_3926 E0M3F8 Pseudomonas sp. CFII64 CFII64_23274 S6GXI3 Pseudomonas synxantha BG33R PseBG33_0275 I4KV50 Pseudomonas syringae pv. actinidiae ICMP 18801 A221_07756 S6XYV3 Pseudomonas syringae pv. actinidiae ICMP 19072 A3SO_07400 S6PNP2 Pseudomonas syringae pv. actinidiae ICMP 19073 A262_20054 S6MLA8 Pseudomonas syringae pv. actinidiae ICMP 19071 A264_07551 S6M2E1 Pseudomonas syringae pv. actinidiae ICMP 19104 A258_19792 S6QSB5 Pseudomonas syringae pv. actinidiae ICMP 9855 A252_19596 S6QRN6 Pseudomonas syringae pv. actinidiae ICMP 19102 A253_19857 S6Q6B9 Pseudomonas syringae pv. actinidiae ICMP 19068 A260_20086 S6Q126 Pseudomonas syringae pv. theae ICMP 3923 A584_21008 S6MKD2 Pseudomonas syringae pv. actinidiae ICMP 19103 A256_19800 S6M4P1 Rhizobium leguminosarum bv. viciae (strain 3841) pRL90300 Q1M8E2 Pseudomonas sp. GM25 PMI24_01694 J2PHH1 Herbaspirillum sp. YR522 PMI40_00700 J3HY53 Pseudomonas syringae pv. morsprunorum str. M302280 PSYMP_05599 F3DS65 Pseudomonas fluorescens (strain Pf0-1) Pfl01_0238 Q3KJS4 Pseudomonas avellanae BPIC 631 Pav631_4731 K2RRZ8 Pseudomonas fluorescens R124 I1A_000262 K0W8U4 Pseudomonas syringae pv. syringae (strain B728a) Psyr_2869 Q4ZSG7 Pseudomonas syringae CC1557 N018_12850 W0MW63 Pseudomonas sp. GM80 PMI37_03766 J3DKC5 Pseudomonas syringae pv. syringae SM PssSM_2902 S3MKC4 Pseudomonas syringae pv. avellanae str. ISPaVe037 Pav037_2494 K2T3F9 Pseudomonas syringae pv. aceris str. M302273 PSYAR_06142 F3JE47 Pseudomonas syringae pv. maculicola str. ES4326 PMA4326_07981 F3HHE2 Pseudomonas syringae BRIP39023 A988_19986 L7GSY0 Pseudomonas syringae pv. aptata str. DSM 50252 PSYAP_18083 F3J2D2 Pseudomonas savastanoi pv. savastanoi NCPPB 3335 PSA3335_0550 D7HUP0 Pseudomonas syringae pv. aesculi str. 0893_23 PSYAE_00125 F3D7S6 Pseudomonas syringae BRIP34881 A987_17762 L7G2P2 Pseudomonas syringae BRIP34876 A979_21556 L7FTL3 Rhizobium leguminosarum bv. viciae WSM1455 Rleg5DRAFT_0033 J0URT9 Pseudomonas syringae Cit 7 PSYCIT7_07619 F3GWQ5 Acinetobacter baumannii NIPH 410 F910_02332 S3TEC4 Acinetobacter baumannii OIFC110 ACIN5110_2029 K5S1X4 Acinetobacter baumannii WC-692 ACINWC692_1619 K1ER91 Pseudomonas sp. TKP U771_01460 V9QPN2 Pseudomonas syringae pv. syringae B64 PssB64_3039 L8NFP3 Pseudomonas syringae pv. actinidiae ICMP 19094 A241_11585 S6VCM5 Pseudomonas syringae pv. actinidiae ICMP 18883 A243_23241 S6TZP7 Pseudomonas syringae pv. actinidiae ICMP 19095 A242_23680 S6TDL4 Pseudomonas syringae pv. actinidiae ICMP 19099 A247_15969 S6S3V9 Pseudomonas syringae pv. actinidiae ICMP 19100 A248_23237 S6R962 Pseudomonas syringae pv. actinidiae ICMP 19098 A246_16023 S6LVQ8

In some embodiments, the disclosure provides methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is a cytochrome P450. In some embodiments, the cytochrome P450 is selected from Table 6 or a variant thereof having at least 90% identity thereto. In some embodiments, the cytochrome P450 is a member of the CYP52 or CYP153 family. In some embodiments, the CYP52 enzyme is selected from CYP52A17, CYP52A13, and CYP52A12.

TABLE 6 Cytochrome P450 enzymes capable of catalyzing selective terminal alkene hydroxylation. Accession Species Origin Gene names No Candida tropicalis (Yeast) CYP52A12 Q874J5 Candida tropicalis (strain ATCC MYA-3404/ CTRG_02725 C5M8K3 T1) (Yeast) Candida tropicalis (Yeast) CYP52A6 P30608 Candida albicans (Yeast) Q9C2X5 Candida maltosa (Yeast) CYP52A3-B P24458 Candida dubliniensis (strain CD36/ATCC CYP52A5 CD36_64140 B9WJ64 MYA-646/CBS 7987/NCPF 3949/NRRL Y- 17841) (Yeast) Candida albicans (strain SC5314/ATCC MYA- ALK1 CaO19.13150 Q5A8M1 2876) (Yeast) orf19.13150 Candida albicans (strain SC5314/ATCC MYA- ALK1 CaO19.5728 Q5A8U5 2876) (Yeast) orf19.5728 Candida maltosa (strain Xu316) (Yeast) G210_4862 M3HRI7 Candida maltosa (Yeast) CYP52A3-A P16496 Candida orthopsilosis (strain 90-125) (Yeast) CORT_0F01930 H8X8E5 Candida parapsilosis (strain CDC 317/ATCC CPAR2_600870 G8B4X9 MYA-4646) (Yeast) (Monilia parapsilosis) Lodderomyces elongisporus (strain ATCC LELG_04957 A5E5R8 11503/CBS 2605/JCM 1781/NBRC 1676/NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida maltosa (Yeast) ALK3-B (CYP52A4) B0VX53 Candida maltosa (Yeast) ALK8-B Q12584 Candida tropicalis (Yeast) CYP52A8 P30610 Debaryomyces hansenii (strain ATCC 36239/ DEHA2E18634g Q6BNV8 CBS 767/JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida tropicalis (Yeast) CYP52A17 Q874I9 Candida maltosa (strain Xu316) (Yeast) G210_3820 M3II00 Spathaspora passalidarum (strain NRRL Y- SPAPADRAFT_59378 G3AJR6 27907/11-Y1) Scheffersomyces stipitis (strain ATCC 58785/ CP52M PICST_58031 A3LRT5 CBS 6054/NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Candida parapsilosis (strain CDC 317/ATCC CPAR2_503950 G8BH23 MYA-4646) (Yeast) (Monilia parapsilosis) Candida parapsilosis (strain CDC 317/ATCC CPAR2_800510 G8BBI4 MYA-4646) (Yeast) (Monilia parapsilosis) Candida tropicalis (Yeast) CYP52A18 Q874I8 Candida maltosa (strain Xu316) (Yeast) G210_4812 M3K5V3 Debaryomyces hansenii (Yeast) (Torulaspora hansenii) CYP52A13 ALK2 Q9Y758 Meyerozyma guilliermondii (strain ATCC 6260/ PGUG_05855 A5DRF4 CBS 566/DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324) (Yeast) (Candida guilliermondii) Debaryomyces hansenii (strain ATCC 36239/ DEHA2C02596g Q6BVH7 CBS 767/JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida maltosa (Yeast) CYP52A5 Q12581 Meyerozyma guilliermondii (strain ATCC 6260/ PGUG_01238 A5DD87 CBS 566/DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324) (Yeast) (Candida guilliermondii) Debaryomyces hansenii (Yeast) (Torulaspora CYP52A12 ALK1 Q9Y757 hansenii) Candida dubliniensis (strain CD36/ATCC CYP52A14 CD36_25250 B9WKL6 MYA-646/CBS 7987/NCPF 3949/NRRL Y-17841) (Yeast) Meyerozyma guilliermondii (strain ATCC 6260/ PGUG_05670 A5DQW9 CBS 566/DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324) (Yeast) (Candida guilliermondii) Candida albicans (strain SC5314/ATCC MYA- ALK2 CaO19.7513 Q5AAH6 2876) (Yeast) orf19.7513 Candida albicans (strain WO-1) (Yeast) CAWG_01382 C4YNC3 Candida tropicalis (Yeast) CYP52A14 CYP14 Q874J3 Candida tropicalis (Yeast) CYP52A13 Q874J4 Pichia sorbitophila (strain ATCC MYA-4447/ Piso0_002820 GNLVRS G8YG24 BCRC 22081/CBS 7064/NBRC 10061/ 01_PISO0I18532g NRRL Y-12695) (Hybrid yeast) Candida parapsilosis (strain CDC 317/ATCC CPAR2_204220 G8BFZ5 MYA-4646) (Yeast) (Monilia parapsilosis) Candida tropicalis (Yeast) CYP52A20 Q874I6 Candida tropicalis (Yeast) CYP52A19 Q874I7 Lodderomyces elongisporus (strain ATCC LELG_00044 A5DRQ8 11503/CBS 2605/JCM 1781/NBRC 1676/ NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida albicans (strain WO-1) (Yeast) CAWG_02011 C4YMD2 Candida albicans (strain SC5314/ATCC MYA- ALK8 CaO19.10 Q59K96 2876) (Yeast) CaO19.7683 Candida albicans (Yeast) alk8 O74626 Candida maltosa (strain Xu316) (Yeast) G210_4811 M3JDC1 Scheffersomyces stipitis (strain ATCC 58785/ CP52C PICST_56580 A3LR60 CBS 6054/NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Lodderomyces elongisporus (strain ATCC LELG_03506 A5E1L9 11503/CBS 2605/JCM 1781/NBRC 1676/ NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida tropicalis (strain ATCC MYA-3404/ CTRG_03115 C5MAM3 T1) (Yeast) Pichia sorbitophila (strain ATCC MYA-4447/ Piso0_002820 GNLVRS G8YDL5 BCRC 22081/CBS 7064/NBRC 10061/ 01_PISO0J20293g NRRL Y-12695) (Hybrid yeast) Candida parapsilosis (strain CDC 317/ATCC CPAR2_204210 G8BFZ4 MYA-4646) (Yeast) (Monilia parapsilosis) Spathaspora passalidarum (strain NRRL Y-27907/11-Y1) SPAPADRAFT_134963 G3AJD3 Candida tropicalis (strain ATCC MYA-3404/T1) (Yeast) CTRG_01061 C5M4S1 Candida tropicalis (Yeast) CYP52A2 P30607 Candida parapsilosis (strain CDC 317/ATCC CPAR2_800520 G8BBI5 MYA-4646) (Yeast) (Monilia parapsilosis) Scheffersomyces stipitis (strain ATCC 58785/ CP52L PICST_56638 A3LSP0 CBS 6054/NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Candida parapsilosis (strain CDC 317/ATCC CPAR2_203780 G8BFV1 MYA-4646) (Yeast) (Monilia parapsilosis) Candida maltosa (strain Xu316) (Yeast) G210_4902 M3IU34 Candida orthopsilosis (strain 90-125) (Yeast) CORT_0D03890 H8X5Y1 Candida dubliniensis (strain CD36/ATCC CD36_32710 B9WMB3 MYA-646/CBS 7987/NCPF 3949/NRRL Y- 17841) (Yeast) Pichia sorbitophila (strain ATCC MYA-4447/ G8YJP0 BCRC 22081/CBS 7064/NBRC 10061/ NRRL Y-12695) (Hybrid yeast) Debaryomyces hansenii (strain ATCC 36239/ DEHA2E18590g Q6BNW0 CBS 767/JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida maltosa (Yeast) CYP52A9 Q12586 Scheffersomyces stipitis (strain ATCC 58785/ ALK2 PICST_35590 A3LS01 CBS 6054/NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Spathaspora passalidarum (strain NRRL Y- SPAPADRAFT_67265 G3APG2 27907/11-Y1) Candida tropicalis (strain ATCC MYA-3404/ CTRG_03120 C5MAM8 T1) (Yeast) Candida maltosa (Yeast) CYP52A11 Q12589 Candida albicans (strain WO-1) (Yeast) CAWG_01383 C4YNC4 Candida tropicalis (strain ATCC MYA-3404/ CTRG_01060 C5M4S0 T1) (Yeast) Candida albicans (strain SC5314/ATCC MYA- ALK3 CaO19.7512 Q5AAH7 2876) (Yeast) orf19.7512 Candida tropicalis (Yeast) CYP52A1 P10615 Scheffersomyces stipitis (strain ATCC 58785/ CYP52 PICST_37142 A3LZV9 CBS 6054/NBRC 10063/NRRL Y-11545) (Yeast) (Pichia stipitis) Debaryomyces hansenii (strain ATCC 36239/ DEHA2E18612g Q6BNV9 CBS 767/JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida tenuis (strain ATCC 10573/BCRC CANTEDRAFT_115474 G3BA51 21748/CBS 615/JCM 9827/NBRC 10315/ NRRL Y-1498/VKM Y-70) (Yeast) Lodderomyces elongisporus (strain ATCC LELG_03309 A5E122 11503/CBS 2605/JCM 1781/NBRC 1676/ NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Lodderomyces elongisporus (strain ATCC LELG_03505 A5E1L8 11503/CBS 2605/JCM 1781/NBRC 1676/ NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida tropicalis (Yeast) CYP52A16 CYP16 Q874J1 Candida tropicalis (Yeast) CYP52A15 Q874J2 Candida maltosa (Yeast) CYP52A10 Q12588 Candida dubliniensis (strain CD36/ATCC ALK3-A CD36_25260 B9WKL7 MYA-646/CBS 7987/NCPF 3949/NRRL Y- 17841) (Yeast) Candida maltosa (Yeast) CYP52A4 P16141 Candida tenuis (strain ATCC 10573/BCRC CANTEDRAFT_113909 G3B3X3 21748/CBS 615/JCM 9827/NBRC 10315/ NRRL Y-1498/VKM Y-70) (Yeast) Meyerozyma guilliermondii (Yeast) (Candida guilliermondii) CYP52 I6UGD5 Spathaspora passalidarum (strain NRRL Y- SPAPADRAFT_153278 G3AMY8 27907/11-Y1) Candida tenuis (strain ATCC 10573/BCRC CANTEDRAFT_116673 G3BEU9 21748/CBS 615/JCM 9827/NBRC 10315/ NRRL Y-1498/VKM Y-70) (Yeast) Candida maltosa (strain Xu316) (Yeast) G210_3821 M3J257 Candida tropicalis (Yeast) CYP52A7 P30609 Clavispora lusitaniae (strain ATCC 42720) CLUG_03984 C4Y750 (Yeast) (Candida lusitaniae) Debaryomyces hansenii (strain ATCC 36239/ DEHA2C01100g Q6BVP2 CBS 767/JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Candida tropicalis (Yeast) CYP52D2 Q874J0 Clavispora lusitaniae (strain ATCC 42720) CLUG_04851 C4Y9G1 (Yeast) (Candida lusitaniae) Meyerozyma guilliermondii (strain ATCC 6260/ PGUG_04005 A5DL54 CBS 566/DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324) (Yeast) (Candida guilliermondii) Yarrowia lipolytica (Candida lipolytica) ALK6 O74132 Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_B01848g F2Z623 (Yeast) (Candida lipolytica) Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_E25982g Q6C4K6 (Yeast) (Candida lipolytica) Yarrowia lipolytica (Candida lipolytica) ALK1 O74127 Yarrowia lipolytica (Candida lipolytica) ALK2 O74128 Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_F01320g F2Z6J3 (Yeast) (Candida lipolytica) Candida maltosa (Yeast) CYP52D1 Q12585 Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_B20702g Q6CDW4 (Yeast) (Candida lipolytica) Byssochlamys spectabilis (strain No. 5/NBRC PVAR5_4403 V5G4E7 109023) (Paecilomyces variotii) Byssochlamys spectabilis (strain No. 5/NBRC PVAR5_4403 V5G4E7 109023) (Paecilomyces variotii) Aspergillus terreus (strain NIH 2624/FGSC A1156) ATEG_02198 Q0CVT6 Neosartorya fischeri (strain ATCC 1020/DSM NFIA_029600 A1D9P7 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) Yarrowia lipolytica (Candida lipolytica) ALK4 O74130 Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_B13816g F2Z6H3 (Yeast) (Candida lipolytica) Penicillium digitatum (strain PHI26/CECT PDIG_58170 K9G9Y0 20796) (Green mold) Penicillium digitatum (strain Pd1/CECT PDIP_67660 K9FGZ9 20795) (Green mold) Aspergillus niger (strain ATCC 1015/CBS ASPNIDRAFT_210944 G3XNK4 113.46/FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) Aspergillus niger (strain CBS 513.88/FGSC An14g01110 A2R2K9 A1513) Tuber melanosporum (strain Mel28) (Perigord GSTUM_00009186001 D5GJT6 black truffle) Yarrowia lipolytica (Candida lipolytica) ALK7 O74133 Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_A15488g F2Z6A4 (Yeast) (Candida lipolytica) Arthrobotrys oligospora (strain ATCC 24927/ AOL_s00109g132 G1XKA3 CBS 115.81/DSM 1491) (Nematode-trapping fungus) (Didymozoophaga oligospora) Dactylellina haptotyla (strain CBS 200.50) H072_6900 S8ADY3 (Nematode-trapping fungus) (Monacrosporium haptotylum) Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_B06248g Q6CFK2 (Yeast) (Candida lipolytica) Aspergillus clavatus (strain ATCC 1007/CBS ACLA_054640 A1C993 513.65/DSM 816/NCTC 3887/NRRL 1) Byssochlamys spectabilis (strain No. 5/NBRC PVAR5_0196 V5FIS1 109023) (Paecilomyces variotii) Aspergillus kawachii (strain NBRC 4308) AKAW_05280 G7XJE1 (White koji mold) (Aspergillus awamori var. kawachi) Aspergillus oryzae (strain 3.042) (Yellow koji mold) Ao3042_00039 I8AC74 Aspergillus flavus (strain ATCC 200026/FGSC AFLA_041790 B8NCU4 A1120/NRRL 3357/JCM 12722/SRRC 167) Aspergillus oryzae (strain ATCC 42149/RIB AO090011000346 Q2U0Q3 40) (Yellow koji mold) Aspergillus oryzae (Yellow koji mold) CYP52H3 D4QC14 Candida tenuis (strain ATCC 10573/BCRC CANTEDRAFT_130130 G3B1J0 21748/CBS 615/JCM 9827/NBRC 10315/ NRRL Y-1498/VKM Y-70) (Yeast) Emericella nidulans (strain FGSC A4/ATCC AN7131.2 ANIA_07131 Q5AX49 38163/CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Talaromyces stipitatus (strain ATCC 10500/ TSTA_012000 B8ME14 CBS 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Starmerella bombicola B8QHP3 Hordeum vulgare var. distichum (Two-rowed F2E8C2 barley) Mycosphaerella graminicola (strain CBS 115943/ CYP- F9X9F0 IPO323) (Speckled leaf blotch fungus) 27 MYCGRDRAFT_70822 (Septoria tritici) Neosartorya fumigata (strain ATCC MYA-4609/ AFUA_4G03800 Q4W9T4 Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus) Neosartorya fumigata (strain CEA10/CBS AFUB_099220 B0YEH7 144.89/FGSC A1163) (Aspergillus fumigatus) Penicillium chrysogenum (strain ATCC 28089/ Pc14g00320 B6H5K4 DSM 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc14g00320 Clavispora lusitaniae (strain ATCC 42720) CLUG_04098 C4Y4W0 (Yeast) (Candida lusitaniae) Penicillium roqueforti CYP52A12PROQFM164_S03g001613 W6QFZ4 Yarrowia lipolytica (Candida lipolytica) ALK5 O74131 Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_B13838g F2Z5W7 (Yeast) (Candida lipolytica) Candida tenuis (strain ATCC 10573/BCRC CANTEDRAFT_107892 G3B8A7 21748/CBS 615/JCM 9827/NBRC 10315/ NRRL Y-1498/VKM Y-70) (Yeast) Penicillium marneffei (strain ATCC 18224/ PMAA_059650 B6QM59 CBS 334.59/QM 7333) Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_A20130g Q6CGD9 (Yeast) (Candida lipolytica) Candida apicola (Yeast) CYP52E2 Q12573 Macrophomina phaseolina (strain MS6) MPH_10814 K2RGW0 (Charcoal rot fungus) Cyphellophora europaea CBS 101466 HMPREF1541_06043 W2RTP6 Cochliobolus sativus (strain ND90Pr/ATCC COCSADRAFT_40532 M2STM3 201652) (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Cochliobolus sativus (strain ND90Pr/ATCC COCSADRAFT_148934 M2SE93 201652) (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Bipolaris victoriae FI3 COCVIDRAFT_89176 W7ES92 Bipolaris zeicola 26-R-13 COCCADRAFT_102357 W6Y6Y4 Cochliobolus heterostrophus (strain C5/ATCC COCHEDRAFT_1085873 M2TJW9 48332/race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Cochliobolus heterostrophus (strain C4/ATCC COCC4DRAFT_201005 N4WTS2 48331/race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Pseudogymnoascus destructans (strain ATCC GMDG_04968 L8GCB9 MYA-4855/20631-21) (Bat white-nose syndrome fungus) (Geomyces destructans) Aspergillus terreus (strain NIH 2624/FGSC A1156) ATEG_03903 Q0CQY1 Marssonina brunnea f. sp. multigermtubi (strain MBM_06876 K1WCN3 MB_m1) (Marssonina leaf spot fungus) Penicillium marneffei (strain ATCC 18224/ PMAA_093890 B6QHD0 CBS 334.59/QM 7333) Neosartorya fumigata (strain CEA10/CBS AFUB_025410 B0XRZ8 144.89/FGSC A1163) (Aspergillus fumigatus) Candida apicola (Yeast) CYP52E1 P43083 Neosartorya fumigata (strain ATCC MYA-4609/ AFUA_2G09540 Q4X1L5 Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus) Neosartorya fischeri (strain ATCC 1020/DSM NFIA_085030 A1DGP3 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) Cordyceps militaris (strain CM01) (Caterpillar CCM_07376 G3JQU8 fungus) Coniosporium apollinis (strain CBS 100218) W97_02755 R7YNR2 (Rock-inhabiting black yeast) Penicillium chrysogenum (strain ATCC 28089/ Pc22g19240 B6HVQ7 DSM 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc22g19240 Penicillium digitatum (strain Pd1/CECT PDIP_65200 K9G3N2 20795) (Green mold) Penicillium digitatum (strain PHI26/CECT PDIG_30820 K9FYP6 20796) (Green mold) Penicillium roqueforti PROQFM164_S01g001598 W6QDZ0 Marssonina brunnea f. sp. multigermtubi (strain MBM_06372 K1WQC3 MB_m1) (Marssonina leaf spot fungus) Botryotinia fuckeliana (strain BcDW1) (Noble BcDW1_3993 M7U1E8 rot fungus) (Botrytis cinerea) Botryotinia fuckeliana (strain T4) (Noble rot BofuT4P27000003001 G2Y6G5 fungus) (Botrytis cinerea) Emericella nidulans (strain FGSC A4/ATCC AN9384.2 ANIA_09384 Q5AQP6 38163/CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Candida maltosa (Yeast) CYP52C2 Q12587 Phaeosphaeria nodorum (strain SN15/ATCC SNOG_02153 Q0V1G1 MYA-4574/FGSC 10173) (Glume blotch fungus) (Septoria nodorum) Pyrenophora tritici-repentis (strain Pt-1C-BFP) PTRG_08257 B2WF96 (Wheat tan spot fungus) (Drechslera tritici-repentis) Pyrenophora teres f. teres (strain 0-1) (Barley net PTT_00451 E3RCI0 blotch fungus) (Drechslera teres f. teres) Aspergillus niger (strain ATCC 1015/CBS ASPNIDRAFT_140405 G3YCS1 113.46/FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) Bipolaris oryzae ATCC 44560 COCMIDRAFT_998 W7A2Q6 Cochliobolus heterostrophus (strain C4/ATCC COCC4DRAFT_169587 N4X0M1 48331/race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Cochliobolus heterostrophus (strain C5/ATCC COCHEDRAFT_1140715 M2U5K5 48332/race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Botryosphaeria parva (strain UCR-NP2) UCRNP2_24 R1GXQ4 (Grapevine canker fungus) (Neofusicoccum parvum) Ajellomyces capsulatus (strain H88) (Darling's HCEG_07709 F0URG7 disease fungus) (Histoplasma capsulatum) Bipolaris oryzae ATCC 44560 COCMIDRAFT_1291 W7A267 Dactylellina haptotyla (strain CBS 200.50) H072_402 S8ARJ7 (Nematode-trapping fungus) (Monacrosporium haptotylum) Cladophialophora carrionii CBS 160.54 G647_04218 V9DES7 Exophiala dermatitidis (strain ATCC 34100/ HMPREF1120_06284 H6C3Q7 CBS 525.76/NIH/UT8656) (Black yeast) (Wangiella dermatitidis) Yarrowia lipolytica (Candida lipolytica) ALK3 O74129 Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_E23474g F2Z6D5 (Yeast) (Candida lipolytica) Blumeria graminis f. sp. hordei (strain DH14) BGHDH14_bgh01926 N1JHB2 (Barley powdery mildew) (Oidium monilioides f. sp. hordei) Neosartorya fischeri (strain ATCC 1020/DSM NFIA_063580 A1D653 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) Dactylellina haptotyla (strain CBS 200.50) H072_3894 S8C337 (Nematode-trapping fungus) (Monacrosporium haptotylum) Aspergillus kawachii (strain NBRC 4308) AKAW_03269 G7XDZ6 (White koji mold) (Aspergillus awamori var. kawachi) Aspergillus niger (strain ATCC 1015/CBS ASPNIDRAFT_183349 G3Y6F0 113.46/FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) Aspergillus oryzae (strain 3.042) (Yellow koji Ao3042_02280 I8IPH3 mold) Aspergillus flavus (strain ATCC 200026/FGSC AFLA_089870 B8NKB3 A1120/NRRL 3357/JCM 12722/SRRC 167) Arthroderma gypseum (strain ATCC MYA-4604/ MGYG_08468 E4V5T0 CBS 118893) (Microsporum gypseum) Arthroderma otae (strain ATCC MYA-4605/ MCYG_06305 C5FUA2 CBS 113480) (Microsporum canis) Bipolaris victoriae FI3 COCVIDRAFT_33397 W7EVM0 Bipolaris oryzae ATCC 44560 COCMIDRAFT_22726 W6ZD79 Byssochlamys spectabilis (strain No. 5/NBRC PVAR5_0072 V5HQF9 109023) (Paecilomyces variotii) Bipolaris zeicola 26-R-13 COCCADRAFT_21391 W6YP58 Mycosphaerella fijiensis (strain CIRAD86) MYCFIDRAFT_153745 M3AEP8 (Black leaf streak disease fungus) (Pseudocercospora fijiensis) Aspergillus terreus (strain NIH 2624/FGSC ATEG_01150 Q0CYT4 A1156) Setosphaeria turcica (strain 28A) (Northern leaf SETTUDRAFT_96298 R0JZR2 blight fungus) (Exserohilum turcicum) Colletotrichum graminicola (strain M1.001/M2/ GLRG_01676 E3Q5P1 FGSC 10212) (Maize anthracnose fungus) (Glomerella graminicola) Aspergillus clavatus (strain ATCC 1007/CBS ACLA_055810 A1C9K9 513.65/DSM 816/NCTC 3887/NRRL 1) Ajellomyces capsulatus (strain G186AR/H82/ HCBG_07070 C0NV90 ATCC MYA-2454/RMSCC 2432) (Darling's disease fungus) (Histoplasma capsulatum) Aspergillus oryzae (strain ATCC 42149/RIB AO090124000014 Q2U799 40) (Yellow koji mold) Aspergillus oryzae (strain ATCC 42149/RIB AO090124000014 Q2U799 40) (Yellow koji mold) Aspergillus niger (strain CBS 513.88/FGSC An11g04220 A2QW84 A1513) Penicillium marneffei (strain ATCC 18224/ PMAA_050330 B6QKF3 CBS 334.59/QM 7333) Tuber melanosporum (strain Mel28) (Perigord GSTUM_00004620001 D5G7M1 black truffle) Colletotrichum higginsianum (strain IMI CH063_01685 H1VAW5 349063) (Crucifer anthracnose fungus) Beauveria bassiana (strain ARSEF 2860) (White BBA_08136 J5JHI3 muscardine disease fungus) (Tritirachium shiotae) Yarrowia lipolytica (strain CLIB 122/E 150) YALI0_C10054g Q6CCE5 (Yeast) (Candida lipolytica) Botryosphaeria parva (strain UCR-NP2) UCRNP2_3112 R1ERB7 (Grapevine canker fungus) (Neofusicoccum parvum) Setosphaeria turcica (strain 28A) (Northern leaf SETTUDRAFT_104406 R0IY48 blight fungus) (Exserohilum turcicum) Aspergillus clavatus (strain ATCC 1007/CBS ACLA_081330 A1CT08 513.65/DSM 816/NCTC 3887/NRRL 1) Mycosphaerella fijiensis (strain CIRAD86) MYCFIDRAFT_65755 M3A1E6 (Black leaf streak disease fungus) (Pseudocercospora fijiensis) Aspergillus oryzae (strain ATCC 42149/RIB AO090026000094 Q2UFS5 40) (Yellow koji mold) Aspergillus oryzae (strain 3.042) (Yellow koji Ao3042_06896 I7ZXQ6 mold) Aspergillus oryzae (Yellow koji mold) CYP584G1 D4QC67 Aspergillus flavus (strain ATCC 200026/FGSC AFLA_138460 B8NGX8 A1120/NRRL 3357/JCM 12722/SRRC 167) Candida tenuis (strain ATCC 10573/BCRC CANTEDRAFT_120218 G3B201 21748/CBS 615/JCM 9827/NBRC 10315/ NRRL Y-1498/VKM Y-70) (Yeast) Aspergillus niger (strain ATCC 1015/CBS ASPNIDRAFT_51356 G3Y8H5 113.46/FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) Aspergillus niger (strain CBS 513.88/FGSC An03g02570 A2QGB4 A1513) Pseudogymnoascus destructans (strain ATCC GMDG_01087 L8FNT2 MYA-4855/20631-21) (Bat white-nose syndrome fungus) (Geomyces destructans) Cladophialophora carrionii CBS 160.54 G647_06237 V9D5I6 Candida albicans (strain WO-1) (Yeast) CAWG_05505 C4YTL0 Coccidioides posadasii (strain RMSCC 757/ CPSG_05074 E9D644 Silveira) (Valley fever fungus) Coccidioides posadasii (strain C735) (Valley CPC735_058630 C5PIZ0 fever fungus) Candida maltosa (strain Xu316) (Yeast) G210_3874 M3J212 Metarhizium acridum (strain CQMa 102) MAC_00168 E9DQZ9 Bipolaris zeicola 26-R-13 COCCADRAFT_112912 W6XW09 Pyronema omphalodes (strain CBS 100304) PCON_11087 U4LQK1 (Pyronema confluens) Bipolaris victoriae FI3 COCVIDRAFT_39745 W7E2W7 Botryotinia fuckeliana (strain T4) (Noble rot BofuT4_P153970.1 G2YW37 fungus) (Botrytis cinerea) Fusarium heterosporum fsdH S0ARX1 Cyphellophora europaea CBS 101466 HMPREF1541_04435 W2RWT0 Metarhizium acridum (strain CQMa 102) MAC_07120 E9EB72 Macrophomina phaseolina (strain MS6) MPH_05063 K2R5H7 (Charcoal rot fungus) Colletotrichum graminicola (strain M1.001/M2/ GLRG_01883 E3Q8M4 FGSC 10212) (Maize anthracnose fungus) (Glomerella graminicola) Bipolaris zeicola 26-R-13 COCCADRAFT_111835 W6Y8G6 Cochliobolus heterostrophus (strain C5/ATCC COCHEDRAFT_1160314 M2SMR0 48332/race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Bipolaris zeicola 26-R-13 COCCADRAFT_101405 W6YJB0 Cochliobolus heterostrophus (strain C4/ATCC COCC4DRAFT_62846 N4XCY6 48331/race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Colletotrichum gloeosporioides (strain Cg-14) CGLO_16193 T0L9W5 (Anthracnose fungus) (Glomerella cingulata) Botryotinia fuckeliana (strain BcDW1) (Noble BcDW1_1090 M7U9F3 rot fungus) (Botrytis cinerea) Botryotinia fuckeliana (strain T4) (Noble rot BofuT4P90000010001 G2YMJ6 fungus) (Botrytis cinerea) Sclerotinia sclerotiorum (strain ATCC 18683/ SS1G_10037 A7EXH2 1980/Ss-1) (White mold) (Whetzelinia sclerotiorum) Penicillium digitatum (strain PHI26/CECT PDIG_44570 K9FT94 20796) (Green mold) Penicillium digitatum (strain Pd1/CECT PDIP_16560 K9GHJ2 20795) (Green mold) Metarhizium anisopliae (strain ARSEF 23/ MAA_06634 E9F2Y5 ATCC MYA-3075) Starmerella bombicola B8QHP1 Penicillium marneffei (strain ATCC 18224/ PMAA_088180 B6QDT4 CBS 334.59/QM 7333) Metarhizium acridum (strain CQMa 102) MAC_09276 E9EHC8 Mycosphaerella pini (strain NZE10/CBS DOTSEDRAFT_74860 N1PCY6 128990) (Red band needle blight fungus) (Dothistroma septosporum) Aspergillus kawachii (strain NBRC 4308) AKAW_10068 G7XYF8 (White koji mold) (Aspergillus awamori var. kawachi) Aspergillus niger (strain CBS 513.88/FGSC An13g03000 A2R1Z6 A1513) Aspergillus niger (strain ATCC 1015/CBS ASPNIDRAFT_44878 G3XQ89 113.46/FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) Beauveria bassiana (strain ARSEF 2860) (White BBA_05173 J4UM22 muscardine disease fungus) (Tritirachium shiotae) Beauveria bassiana (White muscardine disease E2EAF8 fungus) (Tritirachium shiotae) Aspergillus oryzae (strain 3.042) (Yellow koji Ao3042_05622 I8IHV7 mold) Aspergillus flavus (strain ATCC 200026/FGSC AFLA_045270 B8NBF2 A1120/NRRL 3357/JCM 12722/SRRC 167) Aspergillus oryzae (strain ATCC 42149/RIB AO090011000712 Q2TZU9 40) (Yellow koji mold) Aspergillus oryzae (Yellow koji mold) CYP52G3 D4QC12 Endocarpon pusillum (strain Z07020/HMAS-L- EPUS_05482 U1GCZ9 300199) (Lichen-forming fungus) Sclerotinia sclerotiorum (strain ATCC 18683/ SS1G_05980 A7EKY3 1980/Ss-1) (White mold) (Whetzelinia sclerotiorum) Pyrenophora tritici-repentis (strain Pt-1C-BFP) PTRG_06344 B2W8N6 (Wheat tan spot fungus) (Drechslera tritici-repentis) Candida albicans (strain SC5314/ATCC MYA- ALK6 CaO19.13927 Q5AGW4 2876) (Yeast) CaO19.6574 Candida albicans (Yeast) ALK6 CaJ7.0170 G1U9Z0 CaO19.6574 Trichophyton verrucosum (strain HKI 0517) TRV_06353 D4DGP8 Coccidioides immitis (strain RS) (Valley fever CIMG_00331 J3KGS4 fungus) Ajellomyces dermatitidis ATCC 26199 BDFG_02901 T5C2N4 Ajellomyces dermatitidis (strain ATCC 18188/ BDDG_01558 F2T5V8 CBS 674.68) (Blastomyces dermatitidis) Ajellomyces dermatitidis (strain SLH14081) BDBG_07037 C5JWU3 (Blastomyces dermatitidis) Ajellomyces dermatitidis (strain ER-3/ATCC BDCG_07223 C5GSH0 MYA-2586) (Blastomyces dermatitidis) Coccidioides posadasii (strain C735) (Valley CPC735_073410 C5P014 fever fungus) Colletotrichum orbiculare (strain 104-T/ATCC Cob_02045 N4V6W7 96160/CBS 514.97/LARS 414/MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Coccidioides immitis (strain RS) (Valley fever CIMG_11305 J3KDU2 fungus) Talaromyces stipitatus (strain ATCC 10500/ TSTA_056150 B8MRH9 CBS 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Coccidioides posadasii (strain RMSCC 757/ CPSG_06231 E9D8S9 Silveira) (Valley fever fungus) Uncinocarpus reesii (strain UAMH 1704) UREG_01634 C4JJ27 Starmerella bombicola B8QHP5 Pyrenophora tritici-repentis (strain Pt-1C-BFP) PTRG_04241 B2W1A6 (Wheat tan spot fungus) (Drechslera tritici-repentis) Marssonina brunnea f. sp. multigermtubi (strain MBM_07629 K1XPF9 MB_m1) (Marssonina leaf spot fungus) Metarhizium anisopliae (strain ARSEF 23/ MAA_00167 E9EKL9 ATCC MYA-3075) Macrophomina phaseolina (strain MS6) MPH_02135 K2S0W5 (Charcoal rot fungus) Glarea lozoyensis (strain ATCC 20868/ GLAREA_00730 S3CV81 MF5171) Arthroderma otae (strain ATCC MYA-4605/ MCYG_02969 C5FKC8 CBS 113480) (Microsporum canis) Trichophyton verrucosum (strain HKI 0517) TRV_03431 D4D8J5 Hypocrea atroviridis (strain ATCC 20476/IMI TRIATDRAFT_130690 G9P640 206040) (Trichoderma atroviride) Glarea lozoyensis (strain ATCC 74030/ M7I_0305 H0ED06 MF5533) Ajellomyces capsulatus (strain NAm1/WU24) HCAG_08121 A6REQ6 (Darling's disease fungus) (Histoplasma capsulatum) Pyronema omphalodes (strain CBS 100304) PCON_14046 U4LA29 (Pyronema confluens) Endocarpon pusillum (strain Z07020/HMAS-L- EPUS_04540 U1GA45 300199) (Lichen-forming fungus) Penicillium marneffei (strain ATCC 18224/ PMAA_048940 B6QS70 CBS 334.59/QM 7333) Emericella nidulans (strain FGSC A4/ATCC AN6057.2 ANIA_06057 Q5B073 38163/CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Botryotinia fuckeliana (strain T4) (Noble rot BofuT4_P148640.1 G2YX16 fungus) (Botrytis cinerea) Aspergillus terreus (strain NIH 2624/FGSC ATEG_02893 Q0CTU1 A1156) Aspergillus niger (strain ATCC 1015/CBS ASPNIDRAFT_55501 G3XTI8 113.46/FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) Aspergillus kawachii (strain NBRC 4308) AKAW_10154 G7XYN1 (White koji mold) (Aspergillus awamori var. kawachi) Beauveria bassiana (strain ARSEF 2860) (White BBA_02382 J4WEG4 muscardine disease fungus) (Tritirachium shiotae) Sclerotinia sclerotiorum (strain ATCC 18683/ SS1G_13470 A7F790 1980/Ss-1) (White mold) (Whetzelinia sclerotiorum) Beauveria bassiana (strain ARSEF 2860) (White BBA_02428 J5K2V9 muscardine disease fungus) (Tritirachium shiotae) Beauveria bassiana (White muscardine disease E2EAF6 fungus) (Tritirachium shiotae) Cordyceps militaris (strain CM01) (Caterpillar CCM_04719 G3JD19 fungus) Penicillium chrysogenum (strain ATCC 28089/ Pc21g14130 B6HHN6 DSM 1075/Wisconsin 54-1255) (Penicillium PCH_Pc21g14130 notatum) Mycosphaerella pini (strain NZE10/CBS DOTSEDRAFT_70063 N1PRA2 128990) (Red band needle blight fungus) (Dothistroma septosporum) Mycosphaerella pini (strain NZE10/CBS DOTSEDRAFT_70063 N1PRA2 128990) (Red band needle blight fungus) (Dothistroma septosporum) Aspergillus terreus (strain NIH 2624/FGSC ATEG_07540 Q0CFJ4 A1156) Arthroderma benhamiae (strain ATCC MYA- ARB_05989 D4AP22 4681/CBS 112371) (Trichophyton mentagrophytes) Baudoinia compniacensis (strain UAMH 10762) BAUCODRAFT_63612 M2NKX8 (Angels' share fungus) Candida tropicalis (strain ATCC MYA-3404/ CTRG_04959 C5MFW6 T1) (Yeast) Candida tropicalis (Yeast) CYP52C1 P30612 Metarhizium anisopliae (strain ARSEF 23/ MAA_07989 E9F6U0 ATCC MYA-3075) Mycosphaerella graminicola (strain CBS 115943/ CYP- F9XML6 IPO323) (Speckled leaf blotch fungus) 29MYCGRDRAFT_76681 (Septoria tritici) Cladophialophora carrionii CBS 160.54 G647_07950 V9D5N0 Glarea lozoyensis (strain ATCC 20868/ GLAREA_09137 S3DII6 MF5171) Hypocrea virens (strain Gv29-8/FGSC 10586) TRIVIDRAFT_50878 G9N4V4 (Gliocladium virens) (Trichoderma virens) Marssonina brurnnea f. sp. multigermtubi (strain MBM_02308 K1X1G5 MB_m1) (Marssonina leaf spot fungus) Talaromyces stipitatus (strain ATCC 10500/ TSTA_125560 B8MCM6 CBS 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Arthroderma benhamiae (strain ATCC MYA- ARB_01737 D4AZW7 4681/CBS 112371) (Trichophyton mentagrophytes) Colletotrichum higginsianum (strain IMI CH063_01286 H1V527 349063) (Crucifer anthracnose fungus) Trichophyton tonsurans (strain CBS 112818) TESG_03185 F2RW94 (Scalp ringworm fungus) Marssonina brunnea f. sp. multigermtubi (strain MBM_09278 K1WJW9 MB_m1) (Marssonina leaf spot fungus) Aspergillus terreus (strain NIH 2624/FGSC ATEG_06678 Q0CI06 A1156) Claviceps purpurea (strain 20.1) (Ergot fungus) CPUR_01906 M1VZT8 (Sphacelia segetum) Trichophyton rubrum (strain ATCC MYA-4607/ TERG_01394 F2SCB2 CBS 118892) (Athlete's foot fungus) Setosphaeria turcica (strain 28A) (Northern leaf SETTUDRAFT_39981 R0ILM4 blight fungus) (Exserohilum turcicum) Paracoccidioides brasiliensis (strain Pb03) PABG_02000 C0S2T6 Arthroderma gypseum (strain ATCC MYA-4604/ MGYG_08184 E4V596 CBS 118893) (Microsporum gypseum) Trichophyton equinum (strain ATCC MYA- TEQG_06653 F2Q0K1 4606/CBS 127.97) (Horse ringworm fungus) Talaromyces stipitatus (strain ATCC 10500/ TSTA_036210 B8M882 CBS 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Leptosphaeria maculans (strain JN3/isolate LEMA_P030820.1 E4ZWF4 v23.1.3/race Av1-4-5-6-7-8) (Blackleg fungus) (Phoma lingam) Bipolaris victoriae FI3 COCVIDRAFT_43025 W7E4E8 Magnaporthe oryzae (strain Y34) (Rice blast OOU_Y34scaffold00740g4 L7HW63 fungus) (Pyricularia oryzae) Fusarium oxysporum f. sp. cubense (strain race FOC1_g10015382 N4TN76 1) (Panama disease fungus) Fusarium oxysporum f. sp. lycopersici (strain FOXG_00101 J9MB56 4287/CBS 123668/FGSC 9935/NRRL 34936) (Fusarium vascular wilt of tomato) Endocarpon pusillum (strain Z07020/HMAS-L- EPUS_06065 U1GKM5 300199) (Lichen-forming fungus) Sphaerulina musiva (strain SO2202) (Poplar SEPMUDRAFT_55938 N1QLI3 stem canker fungus) (Septoria musiva) Mycosphaerella graminicola (strain CBS 115943/ CYP- F9XH30 IPO323) (Speckled leaf blotch fungus) 31.1MYCGRDRAFT_47046 (Septoria tritici) Penicillium oxalicum (strain 114-2/CGMCC PDE_08994 S8BFY9 5302) (Penicillium decumbens) Mycosphaerella graminicola (strain CBS 115943/ CYP- F9XDL6 IPO323) (Speckled leaf blotch fungus) 30MYCGRDRAFT_73230 (Septoria tritici) Cladophialophora carrionii CBS 160.54 G647_01266 V9DPI5 Togninia minima (strain UCR-PA7) (Esca UCRPA7_6516 R8BF53 disease fungus) (Phaeoacremonium aleophilum) Fusarium oxysporum (strain Fo5176) (Fusarium FOXB_00215 F9F1C9 vascular wilt) Gaeumannomyces graminis var. tritici (strain GGTG_11345 J3PCX6 R3-111a-1) (Wheat and barley take-all root rot fungus) Cochliobolus sativus (strain ND90Pr/ATCC COCSADRAFT_143540 M2R997 201652) (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Neosartorya fumigata (strain CEA10/CBS AFUB_002090 B0XRD5 144.89/FGSC A1163) (Aspergillus fumigatus) Neosartorya fumigata (strain ATCC MYA-4609/ AFUA_1G01690 Q4WKQ1 Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus) Neosartorya fischeri (strain ATCC 1020/DSM NFIA_022940 A1D590 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) Hypocrea atroviridis (strain ATCC 20476/IMI TRIATDRAFT_239723 G9NQ55 206040) (Trichoderma atroviride) Candida orthopsilosis (strain 90-125) (Yeast) CORT_0A06350 H8WY74 Cyphellophora europaea CBS 101466 HMPREF1541_09254 W2SBP3 Penicillium oxalicum (strain 114-2/CGMCC PDE_02656 S8B080 5302) (Penicillium decumbens) Penicillium chrysogenum (strain ATCC 28089/ Pc20g13950 B6HH32 DSM 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc20g13950 Arthroderma gypseum (strain ATCC MYA-4604/ MGYG_07586 E4V3K6 CBS 118893) (Microsporum gypseum) Hypocrea virens (strain Gv29-8/FGSC 10586) TRIVIDRAFT_91340 G9MUE6 (Gliocladium virens) (Trichoderma virens) Botryotinia fuckeliana (strain BcDW1) (Noble BcDW1_3132 M7U3N6 rot fungus) (Botrytis cinerea) Botryosphaeria parva (strain UCR-NP2) UCRNP2_9778 R1E711 (Grapevine canker fungus) (Neofusicoccum parvum) Cochliobolus sativus (strain ND90Pr/ATCC COCSADRAFT_79461 M2SNB8 201652) (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Aspergillus niger (strain CBS 513.88/FGSC An01g00510 A2Q7F5 A1513) Candida dubliniensis (strain CD36/ATCC CD36_71370 B9WK39 MYA-646/CBS 7987/NCPF 3949/NRRL Y-17841) (Yeast) Cochliobolus heterostrophus (strain C4/ATCC COCC4DRAFT_41710 N4XB06 48331/race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Cochliobolus heterostrophus (strain C5/ATCC COCHEDRAFT_1208754 M2VA93 48332/race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Aspergillus clavatus (strain ATCC 1007/CBS ACLA_032820 A1CSC5 513.65/DSM 816/NCTC 3887/NRRL 1) Hypocrea jecorina (strain QM6a) (Trichoderma reesei) TRIREDRAFT_103147 G0R9K0 Trichophyton tonsurans (strain CBS 112818) TESG_02758 F2RVB9 (Scalp ringworm fungus) Glarea lozoyensis (strain ATCC 20868/ GLAREA_12102 S3D2G7 MF5171) Trichophyton rubrum (strain ATCC MYA-4607/ TERG_03231 F2SJM4 CBS 118892) (Athlete's foot fungus) Leptosphaeria maculans (strain JN3/isolate LEMA_P073070.1 E5A7X3 v23.1.3/race Av1-4-5-6-7-8) (Blackleg fungus) (Phoma lingam) Cyphellophora europaea CBS 101466 HMPREF1541_04444 W2RWL1 Hypocrea jecorina (strain QM6a) (Trichoderma reesei) TRIREDRAFT_65036 G0RNX6 Beauveria bassiana (strain ARSEF 2860) (White BBA_09022 J5J6F5 muscardine disease fungus) (Tritirachium shiotae) Cordyceps militaris (strain CM01) (Caterpillar CCM_02084 G3JCK3 fungus) Trichophyton rubrum (strain ATCC MYA-4607/ TERG_05441 F2SSI7 CBS 118892) (Athlete's foot fungus) Botryotinia fuckeliana (strain BcDW1) (Noble BcDW1_9224 M7U6H3 rot fungus) (Botrytis cinerea) Magnaporthe oryzae (strain P131) (Rice blast OOW_P131scaffold01201g5 L7J0M9 fungus) (Pyricularia oryzae) Magnaporthe oryzae (strain Y34) (Rice blast OOU_Y34scaffold00145g13 L7IJZ9 fungus) (Pyricularia oryzae) Magnaporthe oryzae (strain 70-15/ATCC MGG_09920 G4MR75 MYA-4617/FGSC 8958) (Rice blast fungus) (Pyricularia oryzae) Paracoccidioides lutzii (strain ATCC MYA-826/ PAAG_01378 C1GS83 Pb01) (Paracoccidioides brasiliensis) Bipolaris zeicola 26-R-13 COCCADRAFT_9928 W6Y8S8 Verticillium dahliae (strain VdLs.17/ATCC VDAG_04483 G2X2F9 MYA-4575/FGSC 10137) (Verticillium wilt) Trichophyton verrucosum (strain HKI 0517) TRV_02251 D4D581 Arthroderma benhamiae (strain ATCC MYA- ARB_01131 D4AY62 4681/CBS 112371) (Trichophyton mentagrophytes) Chaetomium globosum (strain ATCC 6205/ CHGG_01610 Q2HDU4 CBS 148.51/DSM 1962/NBRC 6347/NRRL 1970) (Soil fungus) Magnaporthe poae (strain ATCC 64411/73-15) M4G6C3 (Kentucky bluegrass fungus) Hypocrea atroviridis (strain ATCC 20476/IMI TRIATDRAFT_45536 G9NQR1 206040) (Trichoderma atroviride) Colletotrichum orbiculare (strain 104-T/ATCC Cob_03064 N4W651 96160/CBS 514.97/LARS 414/MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Penicillium chrysogenum (strain ATCC 28089/ Pc20g11290 B6HG66 DSM 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc20g11290 Ophiocordyceps sinensis (strain Co18/CGMCC OCS_02874 T5AG58 3.14243) (Yarsagumba caterpillar fungus) (Hirsutella sinensis) Pyrenophora teres f. teres (strain 0-1) (Barley net PTT_07245 E3RH76 blotch fungus) (Drechslera teres f. teres) Baudoinia compniacensis (strain UAMH 10762) BAUCODRAFT_71913 M2MX22 (Angels' share fungus) Podospora anserina (strain S/ATCC MYA- PODANS_0_160 B2AFV1 4624/DSM 980/FGSC 10383) (Pleurage anserina) Aspergillus terreus (strain NIH 2624/FGSC ATEG_05807 Q0CKH7 A1156) Hypocrea jecorina (strain QM6a) (Trichoderma reesei) TRIREDRAFT_75713 G0RDE9 Claviceps purpurea (strain 20.1) (Ergot fungus) CPUR_06997 M1WHP2 (Sphacelia segetum) Aspergillus flavus (strain ATCC 200026/FGSC AFLA_128090 B8NNJ8 A1120/NRRL 3357/JCM 12722/SRRC 167) Mycosphaerella fijiensis (strain CIRAD86) MYCFIDRAFT_49209 M3AV82 (Black leaf streak disease fungus) (Pseudocercospora fijiensis) Grosmannia clavigera (strain kw1407/UAMH CMQ_2882 F0XHG6 11150) (Blue stain fungus) (Graphiocladiella clavigera) Lodderomyces elongisporus (strain ATCC LELG_05768 A5H2Q3 11503/CBS 2605/JCM 1781/NBRC 1676/ NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Candida tropicalis (strain ATCC MYA-3404/ CTRG_03114 C5MAM2 T1) (Yeast) Coniosporium apollinis (strain CBS 100218) W97_03898 R7YRY7 (Rock-inhabiting black yeast) Candida parapsilosis (strain CDC 317/ATCC CPAR2_206990 G8BCR1 MYA-4646) (Yeast) (Monilia parapsilosis) Aspergillus niger (strain CBS 513.88/FGSC An02g10700 A5AAH7 A1513) Baudoinia compniacensis (strain UAMH 10762) BAUCODRAFT_187941 M2MV99 (Angels' share fungus) Candida tropicalis (Yeast) CYP52B1 P30611 Aspergillus kawachii (strain NBRC 4308) AKAW_04070 G7XG31 (White koji mold) (Aspergillus awamori var. kawachi) Colletotrichum gloeosporioides (strain Cg-14) CGLO_15455 T0JYY2 (Anthracnose fungus) (Glomerella cingulata) Colletotrichum gloeosporioides (strain Cg-14) CGLO_15455 T0JYY2 (Anthracnose fungus) (Glomerella cingulata) Endocarpon pusillum (strain Z07020/HMAS-L- EPUS_09448 U1HSE1 300199) (Lichen-forming fungus) Arthroderma gypseum (strain ATCC MYA-4604/ MGYG_00140 E5R368 CBS 118893) (Microsporum gypseum) Botryotinia fuckeliana (strain T4) (Noble rot BofuT4_P052870.1 G2XWR8 fungus) (Botrytis cinerea) Exophiala dermatitidis (strain ATCC 34100/ HMPREF1120_00302 H6BMQ6 CBS 525.76/NIH/UT8656) (Black yeast) (Wangiella dermatitidis) Aspergillus oryzae (Yellow koji mold) CYP52K1 D4QC15 Aspergillus oryzae (strain ATCC 42149/RIB AO090010000548 Q2TWI0 40) (Yellow koji mold) Neurospora tetrasperma (strain FGSC 2508/ NEUTE1DRAFT_150004 F8N2K8 ATCC MYA-4615/P0657) Sordaria macrospora (strain ATCC MYA-333/ SMAC_04611 F7W1Z0 DSM 997/K(L3346)/K-hell) Neurospora crassa (strain ATCC 24698/74- NCU09115 Q7S0G0 OR23-1A/CBS 708.71/DSM 1257/FGSC 987) Eutypa lata (strain UCR-EL1) (Grapevine UCREL1_11542 M7T4H1 dieback disease fungus) (Eutypa armeniacae) Neurospora tetrasperma (strain FGSC 2509/ NEUTE2DRAFT_153986 G4U5S9 P0656) Setosphaeria turcica (strain 28A) (Northern leaf SETTUDRAFT_164879 R0JQZ4 blight fungus) (Exserohilum turcicum) Pyrenophora tritici-repentis (strain Pt-1C-BFP) PTRG_11480 B2WN31 (Wheat tan spot fungus) (Drechslera tritici- repentis) Paracoccidioides lutzii (strain ATCC MYA-826/ PAAG_01137 C1GRJ2 Pb01) (Paracoccidioides brasiliensis) Neosartorya fischeri (strain ATCC 1020/DSM NFIA_054110 A1DMP4 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) Sphaerulina musiva (strain SO2202) (Poplar SEPMUDRAFT_149283 M3D461 stem canker fungus) (Septoria musiva) Emericella nidulans (strain FGSC A4/ATCC AN3917.2 ANIA_03917 Q5B6B3 38163/CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Candida orthopsilosis (strain 90-125) (Yeast) CORT_0H01020 H8XAX0 Aspergillus oryzae (strain 3.042) (Yellow koji Ao3042_10143 I8I9N9 mold) Aspergillus flavus (strain ATCC 200026/FGSC AFLA_074560 B8MWJ8 A1120/NRRL 3357/JCM 12722/SRRC 167) Candida parapsilosis (strain CDC 317/ATCC CPAR2_301000 G8B912 MYA-4646) (Yeast) (Monilia parapsilosis) Aspergillus oryzae (strain ATCC 42149/RIB AO090005000220 Q2UT03 40) (Yellow koji mold) Aspergillus oryzae (Yellow koji mold) CYP52G4 D4QC13 Neosartorya fumigata (strain ATCC MYA-4609/ AFUA_9G03090 Q4WD06 Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus) Botryotinia fuckeliana (strain T4) (Noble rot BofuT4_P109530.1 G2Y7G7 fungus) (Botrytis cinerea) Sclerotinia sclerotiorum (strain ATCC 18683/ SS1G_11430 A7F1G0 1980/Ss-1) (White mold) (Whetzelinia sclerotiorum) Pyronema omphalodes (strain CBS 100304) PCON_02181 U4LWN1 (Pyronema confluens) Thielavia heterothallica (strain ATCC 42464/ MYCTH_2294752 G2Q2L5 BCRC 31852/DSM 1799) (Myceliophthora thermophila) Pestalotiopsis fici W106-1 PFICI_00042 W3XJN2 Eutypa lata (strain UCR-EL1) (Grapevine UCREL1_5311 M7TLT9 dieback disease fungus) (Eutypa armeniacae) Colletotrichum orbiculare (strain 104-T/ATCC Cob_03446 N4W590 96160/CBS 514.97/LARS 414/MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Colletotrichum graminicola (strain M1.001/M2/ GLRG_09839 E3QV05 FGSC 10212) (Maize anthracnose fungus) (Glomerella graminicola) Trichophyton verrucosum (strain HKI 0517) TRV_06704 D4DHP8 Sphaerulina musiva (strain SO2202) (Poplar SEPMUDRAFT_39329 M3B7G5 stem canker fungus) (Septoria musiva) Nectria haematococca (strain 77-13-4/ATCC NECHADRAFT_31103 C7YK50 MYA-4622/FGSC 9596/MPVI) (Fusarium solani subsp. pisi) Coniosporium apollinis (strain CBS 100218) W97_03080 R7YPM4 (Rock-inhabiting black yeast) Gaeumannomyces graminis var. tritici (strain GGTG_12245 J3PFH0 R3-111a-1) (Wheat and barley take-all root rot fungus) Fusarium pseudograminearum (strain CS3096) FPSE_11595 K3V5M1 (Wheat and barley crown-rot fungus) Magnaporthe oryzae (strain P131) (Rice blast OOW_P131scaffold00556g2 L7J9P9 fungus) (Pyricularia oryzae) Magnaporthe oryzae (strain Y34) (Rice blast OOU_Y34scaffold00501g3 L7I9Z3 fungus) (Pyricularia oryzae) Magnaporthe oryzae (strain 70-15/ATCC MGG_08956 G4MW35 MYA-4617/FGSC 8958) (Rice blast fungus) (Pyricularia oryzae) Thielavia terrestris (strain ATCC 38088/NRRL THITE_2057357 G2RF28 8126) (Acremonium alabamense) Gibberella fujikuroi (strain CBS 195.34/IMI FFUJ_01480 S0DIN1 58289/NRRL A-6831) (Bakanae and foot rot disease fungus) (Fusarium fujikuroi) Pyronema omphalodes (strain CBS 100304) PCON_09796 U4L3P6 (Pyronema confluens) Gibberella moniliformis (strain M3125/FGSC FVEG_01415 W7LF29 7600) (Maize ear and stalk rot fungus) (Fusarium verticillioides) Magnaporthe oryzae (strain P131) (Rice blast OOW_P131scaffold01216g6 L7IZ69 fungus) (Pyricularia oryzae) Magnaporthe oryzae (strain 70-15/ATCC MGG_08494 G4NAN9 MYA-4617/FGSC 8958) (Rice blast fungus) (Pyricularia oryzae) Fusarium oxysporum f. sp. cubense (strain race FOC4_g10003027 N1RRF1 4) (Panama disease fungus) Chaetomium thermophilum (strain DSM 1495/ CTHT_0057700 G0SCL9 CBS 144.50/IMI 039719) Botryotinia fuckeliana (strain BcDW1) (Noble BcDW1_5818 M7UFT7 rot fungus) (Botrytis cinerea) Verticillium alfalfae (strain VaMs.102/ATCC VDBG_04942 C9SIR0 MYA-4576/FGSC 10136) (Verticillium wilt of alfalfa) (Verticillium albo-atrum) Arthroderma gypseum (strain ATCC MYA-4604/ MGYG_09210 E4V712 CBS 118893) (Microsporum gypseum) Uncinocarpus reesii (strain UAMH 1704) UREG_00942 C4JF41 Bipolaris oryzae ATCC 44560 COCMIDRAFT_34506 W6ZKE3 Paracoccidioides brasiliensis (strain Pb03) PABG_01712 C0S297 Paracoccidioides brasiliensis (strain Pb18) PADG_03693 C1G8V7 Neosartorya fumigata (strain ATCC MYA-4609/ AFUA_6G08460 Q4WMW7 Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus) Neosartorya fumigata (strain CEA10/CBS AFUB_074420 B0Y7N4 144.89/FGSC A1163) (Aspergillus fumigatus) Aspergillus niger (strain ATCC 1015/CBS ASPNIDRAFT_189129 G3XM79 113.46/FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) Coniosporium apollinis (strain CBS 100218) W97_05529 R7YX00 (Rock-inhabiting black yeast) Aspergillus niger (strain CBS 513.88/FGSC An11g07010 A2QWZ5 A1513) Aspergillus niger (strain ATCC 1015/CBS ASPNIDRAFT_56022 G3YAT8 113.46/FGSC A1144/LSHB Ac4/NCTC 3858a/NRRL 328/USDA 3528.7) Neosartorya fischeri (strain ATCC 1020/DSM NFIA_049440 A1DLD3 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) Aspergillus oryzae (strain ATCC 42149/RIB AO090010000075 Q2TXN5 40) (Yellow koji mold) Aspergillus oryzae (strain 3.042) (Yellow koji Ao3042_00404 I8ABC4 mold) Aspergillus oryzae (Yellow koji mold) CYP584E5 D4QC66 Magnaporthe poae (strain ATCC 64411/73-15) M4GA78 (Kentucky bluegrass fungus) Cladophialophora carrionii CBS 160.54 G647_04914 V9DAX0 Podospora anserina (strain S/ATCC MYA- PODANS_1_9520 B2AY12 4624/DSM 980/FGSC 10383) (Pleurage anserina) Gibberella zeae (strain PH-1/ATCC MYA- FG01284.1 FGSG_01284 I1RCH0 4620/FGSC 9075/NRRL 31084) (Wheat head blight fungus) (Fusarium graminearum) Colletotrichum orbiculare (strain 104-T/ATCC Cob_05738 N4VDA5 96160/CBS 514.97/LARS 414/MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Neosartorya fischeri (strain ATCC 1020/DSM NFIA_113870 A1D8Z5 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) Trichophyton verrucosum (strain HKI 0517) TRV_00955 D4D1K4 Botryotinia fuckeliana (strain T4) (Noble rot BofuT4_P140470.1 G2YYT7 fungus) (Botrytis cinerea) Trichophyton rubrum (strain ATCC MYA-4607/ TERG_02747 F2SKM8 CBS 118892) (Athlete's foot fungus) Botryotinia fuckeliana (strain T4) (Noble rot BofuT4_P075800.1 G2XNP1 fungus) (Botrytis cinerea) Setosphaeria turcica (strain 28A) (Northern leaf SETTUDRAFT_159435 R0IYM1 blight fungus) (Exserohilum turcicum) Bipolaris victoriae FI3 COCVIDRAFT_96117 W7ECV3 Bipolaris zeicola 26-R-13 COCCADRAFT_86052 W6YCN0 Podospora anserina (strain S/ATCC MYA- PODANS_3_1920 B2AZX1 4624/DSM 980/FGSC 10383) (Pleurage anserina) Sporothrix schenckii (strain ATCC 58251/de HMPREF1624_01101 U7Q4H5 Perez 2211183) (Rose-picker's disease fungus) Exophiala dermatitidis (strain ATCC 34100/ HMPREF1120_04188 H6BWM7 CBS 525.76/NIH/UT8656) (Black yeast) (Wangiella dermatitidis) Colletotrichum gloeosporioides (strain Cg-14) CGLO_16096 T0JPF3 (Anthracnose fungus) (Glomerella cingulata) Arthroderma benhamiae (strain ATCC MYA- ARB_05099 D4ALA2 4681/CBS 112371) (Trichophyton mentagrophytes) Macrophomina phaseolina (strain MS6) MPH_10488 K2QR42 (Charcoal rot fungus) Trichophyton tonsurans (strain CBS 112818) TESG_05856 F2S4I4 (Scalp ringworm fungus) Trichophyton equinum (strain ATCC MYA- TEQG_04559 F2PUI2 4606/CBS 127.97) (Horse ringworm fungus) Arthroderma benhamiae (strain ATCC MYA- ARB_07892 D4AUH5 4681/CBS 112371) (Trichophyton mentagrophytes) Arthroderma otae (strain ATCC MYA-4605/ MCYG_08648 C5G126 CBS 113480) (Microsporum canis) Aspergillus flavus (strain ATCC 200026/FGSC AFLA_116530 B8NVG6 A1120/NRRL 3357/JCM 12722/SRRC 167) Mycosphaerella graminicola (strain CBS 115943/ CYP- F9XPH9 IPO323) (Speckled leaf blotch fungus) 28MYCGRDRAFT_111399 (Septoria tritici) Penicillium chrysogenum (strain ATCC 28089/ Pc18g04990 B6HBW9 DSM 1075/Wisconsin 54-1255) (Penicillium notatum) PCH_Pc18g04990 Alternaria solani alt2 Q5KTN2 Colletotrichum higginsianum (strain IMI CH063_05380 H1UYS7 349063) (Crucifer anthracnose fungus) Thielavia heterothallica (strain ATCC 42464/ MYCTH_2060315 G2QDC4 BCRC 31852/DSM 1799) (Myceliophthora thermophila) Togninia minima (strain UCR-PA7) (Esca UCRPA7_1480 R8BUP2 disease fungus) (Phaeoacremonium aleophilum) Ophiostoma piceae (strain UAMH 11346) (Sap F503_00556 S3C2T4 stain fungus) Cladophialophora carrionii CBS 160.54 G647_02236 V9DGM2 Botryotinia fuckeliana (strain BcDW1) (Noble BcDW1_141 M7UBZ7 rot fungus) (Botrytis cinerea) Mycobacterium sp. HXN-1500 cyp153 Q65A64 Gordonia amicalis NBRC 100051 = JCM 11271 GOAMI_64_00090 L7L6P4 Mycobacterium austroafricanum B6UKY3 Mycobacterium sp. ENV421 ahpG I7CD96 uncultured bacterium cyp153 W0UDE1 uncultured bacterium P450 Q33DR8 uncultured bacterium P450 Q33DR9 uncultured bacterium cyp153 W0UDG2 uncultured bacterium cyp153 W0UDM1 uncultured bacterium cyp153 W0UCX8 uncultured bacterium cyp153 W0UAP1 uncultured bacterium cyp153 W0UCW9 Polaromonas sp. (strain JS666/ATCC BAA-500) Bpro_5301 Q11ZY2 uncultured bacterium cyp153 W0UDK1 uncultured bacterium cyp153 W0UD29 uncultured bacterium cyp153 W0UD32 uncultured bacterium cyp153 W0UD27 uncultured bacterium cyp153 W0UAW2 uncultured bacterium cyp153 W0UAW6 Parvibaculum sp. S13-6 CYP153A C7A8P8 uncultured bacterium cyp153 W0UDM5 uncultured bacterium cyp153 W0UD31 uncultured bacterium cyp153 W0UDB6 Parvibaculum sp. S13-5 CYP153A C7A8P2 uncultured bacterium P450 Q33DS1 uncultured bacterium cyp153 W0UDK5 uncultured bacterium cyp153 W0UDU1 Tistrella mobilis CYP153A C7A8Q6 uncultured bacterium cyp153 W0UDS7 Parvibaculum sp. S13-6 CYP153A C7A8P9 uncultured bacterium cyp153 W0UB47 Parvibaculum sp. S13-6 CYP153A C7A8P7 gamma proteobacterium S10-1 CYP153A C7A8N2 uncultured bacterium cyp153 W0UDS4 uncultured bacterium cyp153 W0UAY8 uncultured bacterium cyp153 W0UDB2 uncultured bacterium cyp153 W0UB02 uncultured bacterium cyp153 W0UDV5 uncultured bacterium cyp153 W0UDM7 uncultured bacterium cyp153 W0UD83 uncultured bacterium cyp153 W0UD50 Parvibaculum sp. S13-5 CYP153A C7A8P4 Parvibaculum sp. S18-4 CYP153A C7A8S8 Parvibaculum sp. S18-4 CYP153A C7A8S9 uncultured bacterium cyp153 W0UB69 Parvibaculum sp. S13-5 CYP153A C7A8P5 uncultured bacterium cyp153 W0UDU6 uncultured bacterium cyp153 W0UDD0 uncultured bacterium cyp153 W0UDA8 uncultured bacterium cyp153 W0UDC3 uncultured bacterium cyp153 W0UDF5 uncultured bacterium cyp153 W0UDD2 uncultured bacterium cyp153 W0UD99 uncultured bacterium cyp153 W0UB78 uncultured bacterium cyp153 W0UDU2 uncultured bacterium cyp153 W0UD95 uncultured bacterium cyp153 W0UDT1 uncultured bacterium cyp153 W0UD70 uncultured bacterium cyp153 W0UAV3 uncultured bacterium cyp153 W0UDJ0 Parvibaculum sp. S18-4 CYP153A C7A8S7 uncultured bacterium cyp153 W0UD49 uncultured bacterium cyp153 W0UB74 uncultured bacterium cyp153 W0UDG4 uncultured bacterium cyp153 W0UDJ4 uncultured bacterium cyp153 W0UDL1 uncultured bacterium cyp153 W0UD80 uncultured bacterium cyp153 W0UDP8 uncultured bacterium cyp153 W0UDS6 uncultured bacterium cyp153 W0UDC9 uncultured bacterium cyp153 W0UDE6 uncultured bacterium cyp153 W0UDU9 uncultured bacterium cyp153 W0UDC0 uncultured bacterium cyp153 W0UDW1 uncultured bacterium cyp153 W0UDT4 uncultured bacterium cyp153 W0UDB5 uncultured bacterium cyp153 W0UB64 uncultured bacterium cyp153 W0UDA3 uncultured bacterium cyp153 W0UDR7 uncultured bacterium cyp153 W0UB52 uncultured bacterium cyp153 W0UDA5 uncultured bacterium cyp153 W0UDT6 Caulobacter sp. (strain K31) Caul_0020 B0T154 uncultured bacterium cyp153 W0UCV6 uncultured bacterium cyp153 W0UCU1 uncultured bacterium cyp153 W0UDK0 uncultured bacterium cyp153 W0UDI6 uncultured bacterium cyp153 W0UAU9 uncultured bacterium cyp153 W0UAZ2 uncultured bacterium cyp153 W0UD75 uncultured bacterium cyp153 W0UD14 uncultured bacterium cyp153 W0UB97 uncultured bacterium cyp153 W0UD23 uncultured bacterium cyp153 W0UD18 uncultured bacterium cyp153 W0UDQ2 uncultured bacterium cyp153 W0UDH4 uncultured bacterium cyp153 W0UAT6 uncultured bacterium cyp153 W0UD79 uncultured bacterium cyp153 W0UAN4 uncultured bacterium cyp153 W0UDW9 uncultured bacterium cyp153 W0UCZ3 uncultured bacterium cyp153 W0UCZ3 Erythrobacter sp. S11-13 CYP153A C7A8R4 uncultured bacterium cyp153 W0UDK7 Parvibaculum sp. S13-5 CYP153A C7A8P3 uncultured bacterium cyp153 W0UDS2 uncultured bacterium cyp153 W0UD84 uncultured bacterium cyp153 W0UD90 uncultured bacterium cyp153 W0UB38 uncultured bacterium cyp153 W0UCW4 uncultured bacterium cyp153 W0UB22 uncultured bacterium cyp153 W0UDQ8 uncultured Rhizobiales bacterium E0XZ55 HF4000_48A13 uncultured Rhizobiales bacterium E0XZ44 HF4000_48A13 uncultured bacterium P450 Q33DS2 uncultured bacterium P450 Q33DS0 uncultured bacterium cyp153 W0UDB4 Erythrobacter flavus C5MKK1 uncultured bacterium cyp153 W0UD08 uncultured bacterium cyp153 W0UCW2 Sphingobium sp. S13-2 CYP153A C7A8P1 Sphingopyxis sp. S16-14 CYP153A C7A8R8 uncultured bacterium cyp153 W0UD46 Parvibaculum sp. S13-6 CYP153A C7A8P6 uncultured bacterium cyp153 W0UDQ1 uncultured bacterium cyp153 W0UB27 uncultured bacterium cyp153 W0UD73 uncultured bacterium cyp153 W0UDE2 uncultured bacterium cyp153 W0UD17 Erythrobacter sp. S17-1 CYP153A C7A8R9 uncultured bacterium cyp153 W0UD15 uncultured bacterium cyp153 W0UAU6 Erythrobacter flavus CYP153A C7A8N4 uncultured bacterium cyp153 W0UDD6 uncultured bacterium cyp153 W0UDP1 uncultured bacterium cyp153 W0UDF8 uncultured bacterium cyp153 W0UDN8 uncultured bacterium cyp153 W0UDD3 uncultured bacterium cyp153 W0UDN1 uncultured bacterium cyp153 W0UDK3 uncultured bacterium cyp153 W0UD11 uncultured bacterium cyp153 W0UB85 uncultured bacterium cyp153 W0UDI2 Bradyrhizobium sp. CCGE-LA001 BCCGELA001_36078 W1JJD5 uncultured bacterium cyp153 W0UDP5 uncultured bacterium cyp153 W0UB19 uncultured bacterium cyp153 W0UAL6 uncultured bacterium cyp153 W0UDN3 uncultured bacterium cyp153 W0UD72 uncultured bacterium cyp153 W0UCX1 uncultured bacterium cyp153 W0UDF6 uncultured bacterium cyp153 W0UD00 uncultured bacterium cyp153 W0UD65 Caulobacter sp. AP07 PMI01_00728 J2H335 Parvibaculum lavamentivorans (strain DS-1/ Plav_1765 A7HU01 DSM 13023/NCIMB 13966) uncultured bacterium P450 Q33DS3 uncultured bacterium cyp153 W0UDH8 Erythrobacter flavus CYP153A C7A8R2 Erythrobacter sp. S2-1 CYP153A C7A8K9 Erythrobacter citreus CYP153A C7A8R1 Erythrobacter citreus CYP153A C7A8R3 Erythrobacter flavus CYP153A C7A8N5 uncultured bacterium cyp153 W0UD37 Erythrobacter sp. S14-1 CYP153A C7A8Q4 uncultured bacterium cyp153 W0UDF2 uncultured bacterium cyp153 W0UDR6 uncultured bacterium cyp153 W0UAN1 uncultured bacterium cyp153 W0UCX5 uncultured bacterium cyp153 W0UD38 uncultured bacterium cyp153 W0UDM9 uncultured bacterium cyp153 W0UCW7 uncultured bacterium cyp153 W0UB12 uncultured bacterium cyp153 W0UD04 uncultured bacterium cyp153 W0UDQ6 Sphingopyxis macrogoltabida (Sphingomonas ahpG1 Q5F4D9 macrogoltabidus) Afipia broomeae ATCC 49717 HMPREF9695_03199 K8P5Q2 uncultured bacterium cyp153 W0UD96 Parvibaculum sp. S18-4 CYP153A C7A8S5 uncultured bacterium cyp153 W0UAN7 uncultured bacterium cyp153 W0UCS9 uncultured bacterium cyp153 W0UDX6 uncultured bacterium cyp153 W0UDB7 uncultured bacterium cyp153 W0UD56 uncultured bacterium cyp153 W0UD44 Parvibaculum lavamentivorans (strain DS-1/ Plav_2128 A7HV09 DSM 13023/NCIMB 13966) Caulobacter crescentus (strain NA1000/ CCNA_00061 B8GXF2 CB15N) Caulobacter crescentus (strain ATCC 19089/ CC_0063 Q9AC06 CB15) Parvibaculum lavamentivorans (strain DS-1/ Plav_0025 A7HP15 DSM 13023/NCIMB 13966) Caulobacter segnis (strain ATCC 21756/DSM Cseg_0011 D5VDJ3 7131/JCM 7823/NBRC 15250/LMG 17158/ TK0059) (Mycoplana segnis) Novosphingobium sp. PP1Y PP1Y_AT31178 F6IH26 uncultured bacterium cyp153 W0UDC7 uncultured bacterium cyp153 W0UDA2 uncultured bacterium cyp153 W0UDP7 Parvibaculum sp. S18-4 CYP153A C7A8S6 uncultured bacterium cyp153 W0UAK6 uncultured bacterium cyp153 W0UD52 uncultured bacterium cyp153 W0UCU6 uncultured bacterium cyp153 W0UCR4 uncultured bacterium cyp153 W0UCS6 uncultured bacterium cyp153 W0UDV6 uncultured bacterium cyp153 W0UDY0 uncultured bacterium cyp153 W0UDF0 uncultured bacterium cyp153 W0UDF0 uncultured bacterium cyp153 W0UAV7 uncultured bacterium cyp153 W0UDL7 Bradyrhizobium sp. STM 3843 BRAS3843_1530026 H0THQ7 Bradyrhizobium sp. (strain ORS278) BRADO1446 A4YN62 Bradyrhizobium sp. (strain BTAi1/ATCC BBta_6659 A5EQW5 BAA-1182) Caulobacter crescentus OR37 OR37_01714 R0EKG8 Afipia broomeae ATCC 49717 HMPREF9695_03200 K8P2K6 Afipia clevelandensis ATCC 49720 HMPREF9696_02236 K8P5K9 Bradyrhizobiaceae bacterium SG-6C CSIRO_4275 F7QRQ2 Novosphingobium pentaromativorans US6-1 ahpG3 NSU_pLA1167 G6EL94 marine gamma proteobacterium HTCC2143 GP2143_12206 A0YHG8 Sphingopyxis macrogoltabida (Sphingomonas ahpG2 Q5F4D6 macrogoltabidus) uncultured bacterium cyp153 W0UD98 uncultured bacterium cyp153 W0UAZ7 uncultured bacterium cyp153 W0UCU0 uncultured bacterium cyp153 W0UCW6 Bradyrhizobium sp. ORS 375 BRAO375_960079 H0SSR8 Bradyrhizobium sp. ORS 285 BRAO285_1310010 H0RSU1 Bradyrhizobium sp. STM 3809 BRAS3809_1790009 H0SVY3 Rhodopseudomonas palustris (strain BisA53) RPE_4309 Q07IK1 Bradyrhizobium sp. YR681 PMI42_06128 J3CQJ7 Bradyrhizobium sp. STM 3843 BRAS3843_1530027 H0THQ8 Rhodopseudomonas palustris (strain BisB18) RPC_4264 Q20YJ8 Caulobacter sp. (strain K31) Caul_5296 B0T9L7 Sphingopyxis macrogoltabida (Sphingomonas ahpG3 Q5F4D3 macrogoltabidus) Bradyrhizobium oligotrophicum S58 S58_15720 M4ZMZ3 Bradyrhizobium diazoefficiens (strain JCM blr7242 Q89E45 10833/IAM 13628/NBRC 14792/USDA 110) uncultured bacterium cyp153 W0UAK0 uncultured bacterium cyp153 W0UD34 Bradyrhizobium oligotrophicum S58 S58_15730 M4Z3Y5 Erythrobacter litoralis (strain HTCC2594) ELI_14945 Q2N5G0 Erythrobacter sp. SD-21 ED21_32074 A5PDG4 Bradyrhizobium sp. DFCI-1 C207_05440 U1GV14 Bradyrhizobium sp. DFCI-1 C207_05439 U1HA94 Bradyrhizobium diazoefficiens (strain JCM blr7243 Q89E44 10833/IAM 13628/NBRC 14792/USDA 110) Rhodopseudomonas palustris (strain TIE-1) Rpal_1803 B3Q8D0 Bradyrhizobium sp. CCGE-LA001 BCCGELA001_36088 W1JKM5 Parvibaculum lavamentivorans (strain DS-1/ Plav_1782 A7HU17 DSM 13023/NCIMB 13966) Rhodopseudomonas palustris (strain ATCC RPA1613 Q6N9D6 BAA-98/CGA009) Bradyrhizobium sp. S23321 S23_58660 I0GE69 Bradyrhizobium sp. ORS 285 BRAO285_1310011 H0RSU2 Bradyrhizobium sp. ORS 375 BRAO375_960081 H0SSR9 Bradyrhizobium sp. (strain BTAi1/ATCC BBta_6660 A5EQW6 BAA-1182) Bradyrhizobium japonicum USDA 6 BJ6T_79720 G7DEP2 uncultured bacterium cyp153 W0UDA7 uncultured bacterium cyp153 W0UDB9 Afipia sp. P52-10 X566_03415 W3RJ54 Afipia sp. P52-10 X566_20970 W3RG92 marine gamma proteobacterium HTCC2143 GP2143_06774 A0YGV8 Afipia sp. P52-10 X566_16815 W3RJ04 Bradyrhizobium japonicum USDA 6 BJ6T_21500 G7D7D2 Bradyrhizobium sp. WSM471 Bra471DRAFT_06475 H5YKH9 Bradyrhizobium sp. S23321 S23_58670 I0GE70 Rhodopseudomonas palustris (strain DX-1) Rpdx1_3910 E6VIP2 Bradyrhizobium sp. STM 3809 BRAS3809_1790008 H0SVY2 Bradyrhizobium sp. (strain ORS278) BRADO1445 A4YN61 Rhodopseudomonas palustris (strain HaA2) RPB_3934 Q2IT33 Rhodopseudomonas palustris (strain BisB5) RPD_3694 Q132S4 Phenylobacterium zucineum (strain HLK1) p450 PHZ_c0813 B4RGA3 Bradyrhizobium sp. WSM1253 Bra1253DRAFT_03743 I2QGW7 Bradyrhizobium sp. WSM471 Bra471DRAFT_06476 H5YKI0 Bradyrhizobium sp. WSM1253 Bra1253DRAFT_03744 I2QGW8 Bradyrhizobium japonicum USDA 6 BJ6T_21490 G7D7D1 Bradyrhizobium sp. YR681 PMI42_06129 J2WD32 Afipia sp. P52-10 X566_20975 W3RG20 gamma proteobacterium NOR5-3 NOR53_2355 B8KH72 Bradyrhizobium sp. CCGE-LA001 BCCGELA001_12206 W1JZ89 marine gamma proteobacterium HTCC2148 GPB2148_2599 B7RZN8 gamma proteobacterium BDW918 DOK_00120 I2JQ45 Congregibacter litoralis KT71 KT71_14444 A4A7Y2 Bradyrhizobium diazoefficiens (strain JCM blr1853 H7C6Q5 10833/IAM 13628/NBRC 14792/USDA 110) Bradyrhizobium japonicum id311 Q9AND6 uncultured bacterium cyp153 W0UCV0 uncultured bacterium cyp153 W0UAD7 Pseudomonas sp. 19-rlim G3LGZ6 Bradyrhizobium sp. WSM1253 Bra1253DRAFT_06024 I2QN59 Bradyrhizobium sp. WSM471 Bra471DRAFT_01541 H5Y7S1 uncultured gamma proteobacterium E0XZZ2 EB000_65A11 marine gamma proteobacterium HTCC2148 GPB2148_1452 B7RXX8 marine gamma proteobacterium HTCC2143 GP2143_15156 A0Y901 Afipia sp. P52-10 X566_17435 W3RGW1 gamma proteobacterium NOR5-3 NOR53_537 B8KPR5 Glaciecola psychrophila 170 C427_3047 GPSY_3092 K7ADG3 Marinobacter lipolyticus SM19 MARLIPOL_15764 R8AWZ8 gamma proteobacterium IMCC3088 IMCC3088_2432 F3L451 uncultured bacterium P450 Q33DT3 uncultured bacterium P450 Q33DS9 uncultured bacterium P450 Q33DS8 uncultured bacterium cyp153 W0UD71 Congregibacter litoralis KT71 KT71_02837 A4A779 marine gamma proteobacterium HTCC2080 MGP2080_14441 A0Z7J1 Marinobacter santoriniensis NKSG1 MSNKSG1_10343 M7CRK4 Alcanivorax hongdengensis G1C7P2 Alcanivorax sp. DG881 ADG881_2620 B4WXL2 uncultured bacterium P450 Q33DS6 uncultured bacterium cyp153 W0UCP6 uncultured bacterium cyp153 W0UCQ6 Ochrobactrum anthropi CYP153A C7A8M0 uncultured bacterium cyp153 W0UCN8 uncultured bacterium cyp153 W0UCT1 uncultured bacterium cyp153 W0UCT1 uncultured bacterium cyp153 W0UAI3 gamma proteobacterium HIMB55 OMB55_00002070 H3NWG4 Bradyrhizobium sp. DFCI-1 C207_06143 U1H776 gamma proteobacterium HIMB55 OMB55_00014510 H3NWP3 marine gamma proteobacterium HTCC2080 MGP2080_06587 A0Z166 Burkholderia xenovorans (strain LB400) Bxe_A3593 Q143U3 Alcanivorax sp. P2S70 Q670_08165 U7G5C1 Marinobacter hydrocarbonoclasticus ATCC 49840 MARHY3773 H8WA08 Marinobacter sp. EVN1 Q672_10645 U7NYR4 uncultured bacterium P450 Q33DS4 uncultured bacterium cyp153 W0UDA1 uncultured bacterium cyp153 W0UCR5 uncultured bacterium cyp153 W0UD97 uncultured bacterium cyp153 W0UD81 uncultured bacterium cyp153 W0UCN3 uncultured bacterium cyp153 W0UCN5 uncultured bacterium cyp153 W0UCT3 gamma proteobacterium HdN1 ahpG HDN1F_17560 E1VKJ7 Marinobacter adhaerens (strain HP15) HP15_p187g148 E4PSB0 uncultured bacterium P450 Q33DT0 uncultured bacterium P450 Q33DS5 uncultured bacterium cyp153 W0UD61 uncultured bacterium P450 Q33DT1 Alcanivorax hongdengensis B3U002 uncultured bacterium P450 Q33DT2 uncultured bacterium P450 Q33DS7 uncultured bacterium cyp153 W0UCL9 uncultured bacterium cyp153 W0UDB3 Hyphomonas neptunium (strain ATCC 15444) HNE_2042 Q0C0K3 Alcanivorax dieselolei (strain DSM 16502/ ahpG B5T_02075 K0C9X8 CGMCC 1.3690/B-5) Alcanivorax hongdengensis A-11-3 A11A3_15327 L0WAH6 Alcanivorax dieselolei p450 D0Q1H3 Alcanivorax pacificus W11-5 S7S_02138 K2GI89 Marinobacter sp. ES-1 Q666_09590 U7G612 Limnobacter sp. MED105 LMED105_04587 A6GLB5 Marinobacter aquaeolei (strain ATCC 700491/ Maqu_0600 A1TY82 DSM 11845/VT8) (Marinobacter hydrocarbonoclasticus (strain DSM 11845)) Marinobacter sp. EVN1 Q672_13925 U7NUC4 Marinobacter sp. EN3 Q673_05250 U7H5S5 Marinobacter manganoxydans MnI7-9 KYE_03215 G6YPH4 Marinobacter hydrocarbonoclasticus ATCC ahpG2 MARHY2838 H8WCT8 49840 Marinobacter hydrocarbonoclasticus ahpG2 MARHY2838 D9UAS2 (Pseudomonas nautica) Patulibacter medicamentivorans PAI11_40170 H0EAZ2 Acinetobacter baumannii WC-141 ACINWC141_2468 K8ZRD3 Saccharomonospora marina XMU15 SacmaDRAFT_5365 H5X733 Mycobacterium marinum (strain ATCC BAA- cyp153A16 MMAR_3154 B2HGN5 535/M) Mycobacterium abscessus 3A-0930-R p450 MA3A0930R_2169 I9I3J4 Mycobacterium abscessus 3A-0930-S p450 MA3A0930S_1729 I9I1F6 Mycobacterium abscessus 3A-0731 p450 MA3A0731_2042 I9GVU0 Mycobacterium abscessus 3A-0119-R p450 MA3A0119R_2089 I9FPY3 Mycobacterium abscessus 6G-0728-R p450 MA6G0728R_2104 I9DR77 Mycobacterium abscessus subsp. bolletii 1S- p450 MM1S1540310_1492 I9CBZ2 154-0310 Mycobacterium abscessus 6G-0728-5 p450 MA6G0728S_5133 I9A485 Mycobacterium abscessus 3A-0810-R p450 MM3A0810R_2169 I8Q799 Mycobacterium abscessus 3A-0122-S p450 MA3A0122S_1691 I8LTR4 Mycobacterium abscessus 3A-0122-R p450 MA3A0122R_2136 I8L4A4 Mycobacterium abscessus 6G-0212 p450 MA6G0212_2171 I8I9K7 Mycobacterium abscessus subsp. bolletii 1S- p450 MM1S1530915_1484 I8H4G3 153-0915 Mycobacterium abscessus subsp. bolletii 1S- p450 MM1S1520914_2142 I8GFC6 152-0914 Mycobacterium abscessus subsp. bolletii 1S- p450 MM1S1510930_1936 I8G1R8 151-0930 Mycobacterium abscessus 6G-1108 p450 MA6G1108_2106 I8G118 Mycobacterium abscessus 6G-0125-S p450 MA6G0125S_2116 I8F2E6 Mycobacterium abscessus 6G-0125-R p450 MA6G0125R_1143 I8EZ93 Mycobacterium abscessus subsp. bolletii 2B- p450 MM2B0307_1166 I9EQ97 0307 Mycobacterium abscessus subsp. bolletii 2B- p450 MM2B0107_1179 I8Q7R9 0107 Mycobacterium abscessus subsp. bolletii 2B- p450 MM2B1231_1908 I8PT86 1231 Mycobacterium abscessus subsp. bolletii 2B- p450 MM2B0912S_1850 I8KHB7 0912-S Mycobacterium abscessus subsp. bolletii 2B- p450 MM2B0912R_2246 I8JU18 0912-R Mycobacterium abscessus subsp. bolletii 2B- p450 MM2B0626_1842 I8HVB7 0626 uncultured bacterium cyp153 W0UAF0 Parvibaculum lavamentivorans (strain DS-1/ Plav_1951 A7HUI3 DSM 13023/NCIMB 13966) Alcanivorax hongdengensis G1C7L3 Alcanivorax sp. DG881 ADG881_2119 B4X0H6 Marinobacter sp. C1S70 Q667_02605 U7NVJ0 marine gamma proteobacterium HTCC2143 GP2143_06784 A0YGW0 Alcanivorax sp. P2S70 Q670_00635 U7G5B3 Marinobacter goseongensis p450 T1WMH0 gamma proteobacterium BDW918 DOK_13444 I2JHG9 Hirschia baltica (strain ATCC 49814/DSM Hbal_0836 C6XQ13 5838/IFAM 1418) Acinetobacter indicus CIP 110367 P253_02820 V2UD76 Acinetobacter indicus ANC 4215 F956_01111 S3N495 Acinetobacter sp. OC4 cyp Q2MHE2 Acinetobacter baumannii NIPH 527 F921_03852 N9HTE2 Acinetobacter sp. CIP 102129 F973_00680 N8UI43 Acinetobacter sp. NIPH 809 F993_03507 N8P4U1 Acinetobacter baumannii OIFC0162 ACIN5162_A0021 K5DS46 Acinetobacter sp. EB104 nonM Q93SX3 Dietzia cinnamea P4 ES5_05410 E6J787 Acinetobacter sp. WC-743 ACINWC743_A0288 L9LSK8 Acinetobacter baumannii WC-348 ACINWC348_A0080 K9B8A0 Acinetobacter baumannii WC-141 ACINWC141_A0026 K8ZRU7 Acinetobacter baumannii WC-323 ACINWC323_A0095 K9AWS1 Gordonia malaquae NBRC 108250 GM1_050_00120 M3VCF1 Rhodococcus erythropolis SK121 RHOER0001_0266 C3JL15 Acinetobacter sp. COS3 Q674_03885 U7GP11 Acinetobacter guillouiae MSP4-18 L291_2817 S3YTQ7 Acinetobacter gyllenbergii MTCC 11365 L293_2966 S3YIH4 Acinetobacter gyllenbergii CIP 110306 F957_03919 S3MT86 Acinetobacter sp. CIP 110321 F896_03869 R9AJ00 Acinetobacter pittii ANC 3678 F930_03216 N9FYL9 Acinetobacter beijerinckii CIP 110307 F933_03106 N9FFM7 Acinetobacter beijerinckii CIP 110307 F933_03106 N9FFM7 Acinetobacter guillouiae CIP 63.46 F981_00071 N8TRF0 Acinetobacter sp. NIPH 236 F992_00196 N8PQM8 Acinetobacter radioresistens DSM 6976 = ACRAD_64_00110 K6W366 NBRC 102413 = CIP 103788 F939_02890 Acinetobacter sp. NBRC 100985 ACT4_067_00170 G7GIJ8 Williamsia sp. D3 W823_14840 V8CZP3 Rhodococcus ruber BKS 20-38 G352_16177 M2XNX0 Gordonia neofelifaecis NRRL B-59395 SCNU_19987 F1YPY6 Nocardioidaceae bacterium Broad-1 NBCG_04744 E9V105 Rhodococcus erythropolis DN1 N601_30795 T5HW62 Rhodococcus erythropolis (strain PR4/NBRC RER_pREL1-02600 Q3L9B0 100887) Rhodococcus erythropolis DN1 N601_30930 T5HZQ2 Alcanivorax dieselolei B0LCZ6 uncultured bacterium cyp153 W0UD28 uncultured bacterium cyp153 W0UD53 uncultured bacterium cyp153 W0UCL1 Alcanivorax borkumensis ahpG1 Q5K134 Alcanivorax sp. 97CO-5 Y017_09710 W6ZMW5 Alcanivorax borkumensis (strain SK2/ATCC p450 ABO_0201 Q0VM62 700651/DSM 11573) ABO_2288 Alcanivorax borkumensis ahpG2 Q5K133 gamma proteobacterium HIMB55 OMB55_00008700 H3NSZ4 Amycolicicoccus subflavus (strain DSM 45089/ AS9A_4287 F6EL57 DQS3-9A1) Dietzia cinnamea P4 ES5_17094 E6JDU2 Rhodococcus sp. R04 G0YY52 Dietzia sp. DQ12-45-1b L7QFU8 Gordonia terrae C-6 GTC6_22847 R7Y2Z3 Gordonia rubripertincta NBRC 101908 GORBP_030_00030 L7K246 Gordonia polyisoprenivorans NBRC 16320 GOPIP_035_00030 H0RD32 Gordonia amicalis NBRC 100051 = JCM 11271 GOAMI_32_00650 L7L3E0 Nocardia cyriacigeorgica (strain GUH-2) NOCYR_1539 H6R8V9 Mycobacterium gilvum (strain PYR-GCK) Mflv_4592 A4TFM0 (Mycobacterium flavescens (strain ATCC 700033/PYR-GCK)) Acinetobacter sp. ANC 3862 F900_00467 N9M6H3 Rhodococcus erythropolis (strain PR4/NBRC RER_pREL1-02830 Q3L987 100887) Mycobacterium rhodesiae (strain NBB3) MycrhN_5185 G8RXP7 Rhodococcus wratislaviensis IFP 2016 Rwratislav_02222 L2TWM7 Nocardioides sp. CF8 CF8_1774 R7XZ06 Rhodococcus sp. AW25M09 RHODMAR_4781 L8DQ69 Mycobacterium sp. (strain MCS) Mmcs_3218 Q1B709 Mycobacterium sp. (strain JLS) Mjls_3229 A3Q1I0 Mycobacterium sp. (strain KMS) Mkms_3280 A1UI16 Mycobacterium intracellulare MOTT-02 OCO_23030 H8J7G3 Mycobacterium abscessus subsp. bolletii str. GO linC MYCMA_1074 I6ZDN8 06 Mycobacterium abscessus (strain ATCC 19977/ MAB_2048c B1MP79 DSM 44196) Mycobacterium abscessus V06705 M879_18655 T0B128 Mycobacterium abscessus M94 S7W_02670 I0PWL5 Mycobacterium avium subsp. hominissuis 10-4249 O971_10910 V7M646 Mycobacterium parascrofulaceum ATCC BAA- HMPREF0591_1257 D5P513 614 Rhodococcus sp. AW25M09 RHODMAR_0629 L8DBR6 Nocardia asteroides NBRC 15531 NCAST_16_00270 U5E995 Aeromicrobium marinum DSM 15272 HMPREF0063_10876 E2SA86 Mycobacterium abscessus MAB_091912_2446 L833_0535 V6ZS84 Mycobacterium abscessus MAB_082312_2258 L830_0536 V6ZGT1 Mycobacterium abscessus 47J26 MAB47J26_13072 G6X6X6 Nocardioides sp. CF8 CF8_1685 R7XZ92 Gordonia polyisoprenivorans NBRC 16320 GOPIP_007_00470 H0R8L5 Gordonia araii NBRC 100433 GOARA_078_00570 G7H6Y3 marine gamma proteobacterium HTCC2080 MGP2080_13483 A0Z5X9 Gordonia paraffinivorans NBRC 108238 GP2_063_00030 M3V7L0 Planctomyces maris DSM 8797 PM8797T_18726 A6CH25 Amycolicicoccus subflavus (strain DSM 45089/ AS9A_2813 F6EJ28 DQS3-9A1) Candidatus Microthrix parvicella RN1 BN381_420018 R4Z0X4 Gordonia paraffinivorans NBRC 108238 GP2_036_00650 M3TVA1 Nocardioides sp. CF8 CF8_2601 R7XVN2 Mycobacterium chubuense (strain NBB4) Mycch_5830 D2K2F1 Gordonia polyisoprenivorans (strain DSM 44266/ GPOL_c44990 H6MXH6 VH2) Aeromicrobium marinum DSM 15272 HMPREF0063_10264 E2S8A7 Gordonia rubripertincta NBRC 101908 GORBP_109_00410 L7KEM4 Gordonia namibiensis NBRC 108229 GONAM_02_01570 K6XIG5 Gordonia sp. KTR9 KTR9_5380 J9STN3 Gordonia terrae NBRC 100016 GOTRE_050_00060 H5UDF7 Gordonia alkanivorans NBRC 16433 GOALK_030_00300 F9VS44 Gordonia alkanivorans goaBAC B3IX64 Gordonia sp. TF6 aoxA A9CMS7 Alcanivorax borkumensis (strain SK2/ATCC Q6RCE3 700651/DSM 11573) Gordonia malaquae NBRC 108250 GM1_011_00750 M3UVQ9 alpha proteobacterium JLT2015 C725_0051 M2TQQ4 Oceanicola batsensis HTCC2597 OB2597_05915 A3TT18 Sphingobium baderi LL03 L485_17855 T0HGM2 Erythrobacter litoralis (strain HTCC2594) ELI_12445 Q2N6W0 Erythrobacter sp. SD-21 ED21_18817 A5P986 Novosphingobium nitrogenifigens DSM 19370 Y88_2850 F1Z4F0 Sphingopyxis macrogoltabida (Sphingomonas ahpG5 Q5F4D8 macrogoltabidus) Sphingopyxis alaskensis (strain DSM 13593/ Sala_2865 Q1GP52 LMG 18877/RB2256) (Sphingomonas alaskensis) Sphingopyxis macrogoltabida (Sphingomonas ahpG4 Q5F4D1 macrogoltabidus) Novosphingobium aromaticivorans (strain DSM 12444) Saro_0220 Q2GBV5 Dickeya dadantii (strain Ech586) Dd586_1369 D2BW78 Sphingopyxis sp. MC1 EBMC1_05939 N9UVB0 Dietzia sp. D5 W0C650 Sphingobium indicum B90A SIDU_06697 I5BFE4 Sphingobium chinhatense IP26 M527_09955 W1KG42 Sphingobium sp. HDIP04 L286_21540 T0G3B9 Erythrobacter sp. NAP1 NAP1_13673 A3WFL2 Dickeya dadantii (strain 3937) (Erwinia Dda3937_03358 E0SIQ2 chrysanthemi (strain 3937)) Sphingomonas sanxanigenens DSM 19645 = NX02 NX02_10200 W0AB84 Sphingopyxis sp. MC1 EBMC1_03994 N9WE44 Dickeya sp. D s0432-1 A544_2711 U6Z9W7 Novosphingobium aromaticivorans (strain DSM 12444) Saro_1821 Q2G7B2 Erythrobacter litoralis (strain HTCC2594) ELI_09815 Q2N8D6 Parvibaculum lavamentivorans (strain DS-1/ Plav_0029 A7HP19 DSM 13023/NCIMB 13966) Novosphingobium pentaromativorans US6-1 NSU_3817 G6EHJ6

In some embodiments, the disclosure provide methods for synthesizing olefinic alcohol products as described above, wherein the enzyme is selected from AlkB, AlkB P1, and AlkB1 AB. In some embodiments, the enzyme is selected from CYP153 M. sp.; CYP153A M. aq.; CYP153A M. aq. (G307A); Cyp153A M. aq. (G307A)-CPR_(BM3); Cyp153A P.sp.-CPR_(BM3); CYP153A13N2; CYP153A13N3; CYP153A13P2; and CYP153A7. In some embodiments, the enzyme is selected from CYP52A13 and CYP52A3.

In a related aspect, the disclosure provides a whole cell catalyst comprising an enzyme capable of selectively hydroxylating one terminal carbon of an unsaturated or saturated hydrocarbon substrate. In some embodiments, the cell is a microbial cell. In some embodiments, the enzyme is selected from the group consisting of a non-heme diiron monooxygenase, a long-chain alkane hydroxylase, a cytochrome P450, and combinations thereof. In some embodiments, the enzyme is selected from Table 4, Table 5, Table 6, or a variant thereof having at least 90% identity thereto.

TABLE 6 Exemplary strains suitable for the present disclosure. Plasmid Genotype (or relevant gene Species deletions) Reference E. coli K12 GEc137 pGEc47J contains alkBFGKL alkST Grant et al. Enzyme Microb. Technol. 2011 E. coli W3110 pBT10 contains alkBFG alkST Schrewe et al. Adv. Synth. Cat. 2011 E. coli W3110 pBTL10 contains alkBFGL alkST Julsing et al. Adv. Synth. Cat. 2011 E. coli BL21(DE3) pET-28a(+)-LadA contains LadA Dong et al. Appl. Microbiol. Biotechnol. 2012 E. coli BL21(DE3) pET-28a(+)-CYP153A6 operon Gudimichi et al. Appl. Microbiol. Biotechnol. 2012 E. coli JM109 pJOE-CYP153A_(M.aq.)(G307A)-CPR_(BM3) Scheps et al. Microb. E. coli HMS174 pET-28(+)-CYP153A_(M.aq.)(G307A)-CPR_(BM3) Biotechnol. 2013 E. coli HMS174 pColaDuet-1-CYP153A_(M.aq.)(G307A)- CPR_(BM3), alkL E. coli HMS174 pET-28(+)-CYP153A_(P. sp.)-CPR_(BM3) Malca el al. Chem. Comm. 2012 C. tropicalis DP522 DP1 Δcyp52a17/Δ cyp52a18 Δ Lu et al. J. Am. cyp52a13/Δcyp52a14 Δ fao1/Δ fao1b Δ Chem. Soc. 2010 fao2a/Δ fao2b Δcyp52a12/Δcyp52a12b Δadh-a4/Δadh-a4b Δadhb4/Δadh-b4b Δadh- a10 Δ adh-b11 pXICL::CYP52A13 C. tropicalis DP526 DP1 Δcyp52a17/Δ cyp52a18 Δ cyp52a13/Δcyp52a14 Δ fao1/Δ fao1b Δ fao2a/Δ fao2b Δcyp52a12/Δcyp52a12b Δadh-a4/Δadh-a4b Δadhb4/Δadh-b4b Δadh- a10 Δ adh-b11 pXICL::CYP52A12 C. tropicalis DP428 DP1 Δcyp52a17/Δ cyp52a18 Δ cyp52a13/Δcyp52a14 Δ fao1/Δ fao1b Δ fao2a/Δ fao2b Δcyp52a12/Δcyp52a12b Δadh-a4/Δadh-a4b Δadhb4/Δadh-b4b Δadh- a10 Δ adh-b11 pXICL::CYP52A17

The methods of the disclosure allow for the production of terminal alcohols with controlled regioselectivity, while disfavoring the formation of unwanted species such as epoxides or elimination products. The stereochemistry of an olefinic alcohol product will depend on factors including the structure of the particular olefinic substrate used in a particular reaction, as well as the identity of the enzyme. The methods of the disclosure can be conducted with enzymes that are selective for particular substrates (e.g., cis or Z alkenes vs. trans or E alkenes), as well as with enzymes that demonstrate terminal selectivity (e.g., hydroxylation of one end of an asymmetric alkene vs. the other end of the asymmetric alkene).

In certain instances, a hydroxylase enzyme will exhibit catalytic efficiency with one isomer of an internal alkene (e.g., the cis or Z isomer of an internal alkene) that is greater than the catalytic efficiency exhibited with the other isomer of the same internal alkene (e.g., the trans or E isomer of an internal alkene). In some embodiments, the disclosure provides methods wherein the catalytic efficiency of the hydroxylase enzyme is at least about 2-fold greater with one isomer of an internal alkene than with the other isomer of the internal alkene. The catalytic efficiency exhibited by a hydroxylase with one isomer of an internal alkene can be, for example, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or at least about 500-fold greater than the catalytic efficiency exhibited by the hydroxylase with the other isomer of the internal alkene.

A particular enzyme can therefore produce Z product over E product from a mixture of Z and E isomeric substrates or enrich the Z product over the E product. In certain embodiments, the disclosure provides methods for preparing olefinic alcohol products wherein the Z:E (cis:trans) isomeric ratio of the olefinic alcohol product is different from the Z:E (cis:trans) isomeric ratio of the olefinic substrate. The Z:E isomeric ratio of the olefinic alcohol product can be, for example, around 2 times greater than the Z:E isomeric ratio of the olefinic substrate. The Z:E isomeric ratio of the olefinic alcohol product can be, for example, around 1.25 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, or 40 times greater than the Z:E isomeric ratio of the olefinic substrate.

In some embodiments, the disclosure provides methods for preparing olefinic alcohol products wherein the E:Z (trans:cis) isomeric ratio of the olefinic alcohol product is different from the E:Z (trans:cis) isomeric ratio of the olefinic substrate. The E:Z isomeric ratio of the olefinic alcohol product can be, for example, around 2 times greater than the E:Z isomeric ratio of the olefinic substrate. The E:Z isomeric ratio of the olefinic alcohol product can be, for example, around 1.25 times, 1.5 times, 2 times, 2.5 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, or 40 times greater than the E:Z isomeric ratio of the olefinic substrate.

In some embodiments, the Z:E isomeric ratio of the olefinic alcohol is about 1.25 times greater than the Z:E isomeric ratio of the olefinic substrate. In some embodiments, the E:Z isomeric ratio of the olefinic alcohol is about 1.25 times greater than the E:Z isomeric ratio of the olefinic substrate.

In certain instances, the biohydroxylation reactions in the methods of the disclosure have the potential to form a mixture of two or more products from the same substrate. When an olefinic substrate is asymmetric, for example, hydroxylation of one end/terminus of the substrate leads to one product while hydroxylation of the other end/terminus of the substrate leads to a different product. A reaction could therefore result in a mixture of two olefinic alcohol products. The terminal isomer ratio of an asymmetric olefinic alcohol product can range from about 1:99 to about 99:1. The terminal isomer ratio can be, for example, from about 1:99 to about 1:75, or from about 1:75 to about 1:50, or from about 1:50 to about 1:25, or from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The terminal isomer ratio can be from about 1:80 to about 1:20, or from about 1:60 to about 1:40, or from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The terminal isomer ratio can be about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, or about 1:95. The terminal isomer ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.

The distribution of a product mixture can be expressed as a regioselectivity percentage (“regioselectivity %”). Taking the reaction in FIG. 3 as a non-limiting example, for instance, the regioselectivity of (Z)-5-hexadecene hydroxylation can be calculated using the formula: regioselectivity %=[(χ₁₁)/(χ₁₁+χ₅)]×100%, wherein χ₁₁ is the mole fraction for (Z)-11-hexadecen-1-ol and wherein χ₅ is the mole fraction for (Z)-5-hexadecen-1-ol. In general, the regioselectivity % with respect to terminal alcohol isomers ranges from about 1% to about 99%. The regioselectivity % can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The regioselectivity % can be at least about 5% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

In some embodiments, the regioselectivity % is at least about 60%. In some embodiments, the regioselectivity % is at least about 60% and the Z:E isomeric ratio of the olefinic alcohol is about 1.25 times greater than the Z:E isomeric ratio of the olefinic substrate.

In certain instances, varying levels of olefin epoxidation will occur during the biohydroxylation reactions used in the methods of the disclosure. See, e.g., Scheme 7. Epoxidation of terminal alkenes, in particular, can occur when certain hydroxylase enzymes are used. It is often desirable to minimize such epoxidation or avoid the formation of epoxides altogether. Typically, methods of the disclosure are conducted with hydroxylase enzymes that produce product mixtures with alcohol product:epoxide ratios of at least 1:1. The alcohol product:epoxide ratio can range from about 1:1 to about 99:1. The alcohol:epoxide ratio can be, for example, from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The alcohol:epoxide ratio can be from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The alcohol:epoxide ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.

In some embodiments, methods are conducted using an enzyme that produces an olefinic alcohol product:epoxide product ratio of greater than 1:1. In some embodiments, the enzyme produces an olefinic alcohol product:epoxide product ratio of greater than 2:1.

The distribution of a product mixture can be expressed as a percent selectivity for hydroxylation vs. epoxidation. Taking the reaction in Scheme 7a as a non-limiting example, the percent selectivity for hydroxylation vs. epoxidation of a terminal alkene can be calculated using the formula: selectivity %=[(χ_(H))/(χ_(H)+χ_(E))]×100%, wherein χ_(H) is the mole fraction for the hydroxylation product (i.e., the terminal olefinic alcohol) and wherein χ_(E) is the mole fraction for the epoxidation product (i.e., the terminal epoxide). In general, the percent selectivity for hydroxylation vs. epoxidation ranges from about 1% to about 99%. The percent selectivity for hydroxylation vs. epoxidation can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The percent selectivity for hydroxylation vs. epoxidation can be about 5% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

When halogen-substituted substrates are used in the methods of the disclosure, varying levels of dehalogenation can occur during hydroxylation. Dehalogenation typically results in the formation of aldehyde byproduct. Preferably, dehalogenation is minimized or avoided during the hydroxylation reactions. Typically, methods of the disclosure are conducted with hydroxylase enzymes that produce product mixtures with alcohol:aldehyde ratios of at least 1:1. The alcohol:aldehyde ratio of the product can range from about 1:1 to about 99:1. The alcohol:aldehyde ratio can be, for example, from about 99:1 to about 75:1, or from about 75:1 to about 50:1, or from about 50:1 to about 25:1. The alcohol:aldehyde ratio can be from about 80:1 to about 20:1 or from about 60:1 to about 40:1. The alcohol:aldehyde ratio can be about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, or about 95:1.

The distribution of a product mixture can be expressed as a percent selectivity for hydroxylation vs. dehalogenation. The percent selectivity for hydroxylation vs. dehalogenation of a halogen-substituted substrate can be calculated using the formula: selectivity %=[(χ_(H))/(χ_(H)+χ_(A))]×100%, wherein χ_(H) is the mole fraction for the hydroxylation product and wherein χ_(A) is the mole fraction for the aldehyde product. In general, the percent selectivity for hydroxylation vs. dehalogenation ranges from about 1% to about 99%. The percent selectivity for hydroxylation vs. dehalogenation can be from about 1% to about 99%, or from about 20% to about 80%, or from about 40% to about 60%, or from about 1% to about 25%, or from about 25% to about 50%, or from about 50% to about 75%. The percent selectivity for hydroxylation vs. dehalogenation can be about 5% 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

Biohydroxylation of Subterminal Carbon

In some embodiments, biohydroxylation can occur on an subterminal carbon as shown in Scheme 8. Accordingly, in some embodiments, the disclosure provides for isomers of a sex pheromone which include an subterminal alcohol functional group, an subterminal acetyl, or an subterminal ketone, provided, however, that the subterminal ketone is not located on the same carbon that forms a double bond with an adjacent carbon.

In some embodiments, for example, n is 0, m is 1, i is 3; or n is 1, m is 1, and i is 2; or n is 2, m is 1, and i is 1; or n is 0, m is 2, i is 2; or n is 1, m is 2, and i is 1; or n is 0, m is 3, i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 4; or n is 1, m is 1; and i is 3; or n is 2, m is 1, and i is 2; or n is 3, m is 1, and i is 1; n is 0, m is 2, i is 3; or n is 1, m is 2, and i is 2; or n is 2, m is 2, and i is 1; n is 0, nm is 3, i is 2; or n is 1, m is 3, and i is 1; n is 0, m is 4, i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 5; or n is 1, m is 1, and i is 4; or n is 2, m is 1, and i is 3; or n is 3, m is 1, i is 2; or n is 4, m is 1, and i is 1; or n is 0, m is 2, and i is 4; or n is 1, m is 2, and i is 3; or n is 2, m is 2, and i is 2; or n is 3, m is 2; and i is 1; or n is 0, m is 3, and i is 3; or n is 1, n is 3 and i is 2; or n is 2, m is 3, and i is 1; or n is 0, m is 4, and i is 2; or n is 1, m is 4 and i is 1; or n is 0, m is 5, and i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1 and i is 6; or n is 1, m is 1, and i is 5; or n is 2, m is 1, and i is 4; or n is 3, m is 1, and i is 3; or n is 4, m is 1; and i is 2; or n is 5, m is 1, and i is 1; or n is 0, m is 2 and i is 5; or n is 1, m is 2, i and is 4; or n is 2, m is 2, and i is 3; or n is 3, m is 2, and i is 2; or n is 4, m is 2, and i is 1; or n is 0, m is 3 and i is 4; or n is 1, m is 3, i is 3; or n is 2, m is 3, and i is 2; or n is 3, m is 3, and i is 1; or n is 0, m is 4 and i is 3; or n is 1, m is 4, i is 2; or n is 2, m is 4, and i is 1; or n is 0, m is 5 and i is 2; or n is 1, m is 5, i is 1; or n is 0, m is 6, and i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 7; or n is 1 and m is 1, and i is 6; or n is 2, m is 1, and i is 5; or n is 3, m is 1, and i is 4; or n is 4, m is 1, and i is 3; or n is 5, m is 1, and i is 2; or n is 6, m is 1; and i is 1; n is 0, m is 2, and i is 6; or n is 1 and m is 2, and i is 5; or n is 2, m is 2, and i is 4; or n is 3, m is 2, and i is 3; or n is 4, m is 2, and i is 2; or n is 5, m is 2, and i is 1; n is 0, m is 3, and i is 5; or n is 1, and m is 3, and i is 4; or n is 2, m is 3, and i is 3; or n is 3, m is 3, and i is 2; or n is 4, m is 3, and i is 1; n is 0, m is 4, and i is 4; or n is 1 and m is 4, and i is 3; or n is 2, m is 4, and i is 2; or n is 3, m is 4, and i is 1; n is 0, m is 5, and i is 3; or n is 1 and m is 5, and i is 2; or n is 2, m is 5, and i is 1; n is 0, m is 6, and i is 2; or n is 1 and m is 6, and i is 1; n is 0, m is 7, and i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1; and i is 8; or n is 1, m is 1, and i is 7; or n is 2, m is 1, i is 6; or n is 3, m is 1, i is 5; or n is 4, m is 1, i is 4; or n is 5, m is 1, i is 3; or n is 6, m is 1, i is 2 or n is 7, m is 1, and i is 1; n is 0, m is 2; and i is 7; or n is 1, m is 2, and i is 6; or n is 2, m is 2, i is 5; or n is 3, m is 2, i is 4; or n is 4, m is 2, i is 3; or n is 5, m is 2, i is 2; or n is 6, m is 2, i is 1; n is 0, m is 3; and i is 6; or n is 1, m is 3, and i is 5; or n is 2, m is 3, i is 4; or n is 3, m is 3, i is 3; or n is 4, m is 3, i is 2; or n is 5, m is 3, i is 1; n is 0, m is 4; and i is 5; or n is 1, m is 4, and i is 4; or n is 2, m is 4, i is 3; or n is 3, m is 4, i is 2; or n is 4, m is 4, i is 1; n is 0, m is 5; and i is 4; or n is 1, m is 5, and i is 3; or n is 2, m is 5, i is 2; or n is 3, m is 5, i is 1; n is 0, m is 6; and i is 3; or n is 1, m is 6, and i is 2; or n is 2, m is 6, i is 1; n is 0, m is 7; and i is 2; or n is 1, m is 7, and i is 1; or n is 0, m is 8, i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 9; or n is 1, m is 1, i is 8; or n is 2, m is 1, and i is 7; or n is 3, m is 1, and i is 6; or n is 4, m is 1, and i is 5; or n is 5, m is 1, and i is 4; or n is 6, m is 1, and i is 3; or n is 7, m is 1, and i is 2; or n is 8, m is 1 and i is 1; n is 0, m is 2, and i is 8; or n is 1, m is 2, i is 7; or n is 2, m is 2, and i is 6; or n is 3, m is 2, and i is 5; or n is 4, m is 2, and i is 4; or n is 5, m is 2, and i is 3; or n is 6, m is 2, and i is 2; or n is 7, m is 2, and i is 1; n is 0, m is 3, and i is 7; or n is 1, m is 3, i is 6; or n is 2, m is 3, and i is 5; or n is 3, m is 3, and i is 4; or n is 4, m is 3, and i is 3; or n is 5, m is 3, and i is 2; or n is 6, m is 3, and i is 1; n is 0, m is 4, and i is 6; or n is 1, m is 4, i is 5; or n is 2, m is 4, and i is 4; or n is 3, m is 4, and i is 3; or n is 4, m is 4, and i is 2; or n is 5, m is 4, and i is 1; n is 0, m is 5, and i is 5; or n is 1, m is 5, i is 4; or n is 2, m is 5, and i is 3; or n is 3, m is 5, and i is 2; or n is 4, m is 5, and i is 1; n is 0, m is 6, and i is 4; or n is 1, m is 6, i is 3; or n is 2, m is 6, and i is 2; or n is 3, m is 6, and i is 1; n is 0, m is 7, and i is 3; or n is 1, m is 7, i is 2; or n is 2, m is 7, and i is 1; n is 0, m is 8, and i is 2; or n is 1, m is 8, i is 1; n is 0, n is 9, and i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 10; or n is 1, m is 1, and i is 9; or n is 2, m is 1, and i is 8; or n is 3, m is 1, and i is 7; or n is 4, m is 1, and i is 6; or n is 5, n is 1, i is 5; or n is 6, m is 1, and i is 4; or n is 7, m is 1 and i is 3; or n is 8, m is 1, i is 2; or n is 9, m is 1, and i is 1; or n is 0, m is 2, and i is 9; or n is 1, m is 2, and i is 8; or n is 2, m is 2, and i is 7; or n is 3, m is 2, and i is 6; or n is 4, m is 2, and i is 5; or n is 5, m is 2, i is 4; or n is 6, m is 2, and i is 3; or n is 7, m is 2 and i is 2; or n is 8, m is 2, i is 1; n is 0, m is 3, and i is 8; or n is 1, m is 3, and i is 7; or n is 2, m is 3, and i is 6; or n is 3, m is 3, and i is 5; or n is 4, m is 3, and i is 4; or n is 5, m is 3, i is 3; or n is 6, m is 3, and i is 2; or n is 7, m is 3 and i is 1; n is 0, m is 4, and i is 7; or n is 1, m is 4, and i is 6; or n is 2, m is 4, and i is 5; or n is 3, m is 4, and i is 4; or n is 4, m is 4, and i is 3; or n is 5, m is 4, i is 2; or n is 6, m is 4, and i is 1; n is 0, m is 5, and i is 6; or n is 1, m is 5, and i is 5; or n is 2, m is 5, and i is 4; or n is 3, m is 5, and i is 3; or n is 4, m is 5, and i is 2; or n is 5, m is 5, i is 1; n is 0, m is 6, and i is 5; or n is 1, m is 6, and i is 4; or n is 2, m is 6, and i is 3; or n is 3, m is 6, and i is 2; or n is 4, m is 6, and i is 1; n is 0, m is 7, and i is 4; or n is 1, m is 7, and i is 3; or n is 2, m is 7, and i is 2; or n is 3, m is 7, and i is 1; n is 0, m is 8, and i is 3; or n is 1, m is 8, and i is 2; or n is 2, m is 8, and i is 1; n is 0, m is 7, and i is 4; or n is 1, m is 7, and i is 3; or n is 2, m is 7, and i is 2; or n is 3, m is 7, and i is 1; n is 0, m is 9, and i is 2; or n is 1, m is 9, and i is 1; or n is 0, m is 10, and i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 11; or n is 1, m is 1, and i is 10; or n is 2, m is 1, and i is 9; or n is 3, m is 1, and i is 8; or n is 4, m is 1, and i is 7; or n is 5, m is 1, and i is 6; or n is 6, m is 1 and i is 5; or n is 7, m is 1 and i is 4; or n is 8, m is 1, and i is 3; or n is 9, m is 1, and i is 2; or n is 10, m is 1 and i is 1; n is 0, m is 2, and i is 10; or n is 1, m is 2, and i is 9; or n is 2, m is 2, and i is 8; or n is 3, m is 2, and i is 7; or n is 4, m is 2, and i is 6; or n is 5, m is 2, and i is 5; or n is 6, m is 2 and i is 4; or n is 7, m is 2 and i is 3; or n is 8, m is 2, and i is 2; or n is 9, m is 2 and i is 1; n is 0, m is 3, and i is 9; or n is 1, m is 3, and i is 8; or n is 2, m is 3, and i is 7; or n is 3, m is 3, and i is 6; or n is 4, m is 3, and i is 5; or n is 5, m is 3, and i is 4; or n is 6, m is 3 and i is 3; or n is 7, m is 3 and i is 2; or n is 8, m is 3, and i is 1; n is 0, m is 4, and i is 8; or n is 1, m is 4, and i is 7; or n is 2, m is 4, and i is 6; or n is 3, m is 4, and i is 5; or n is 4, m is 4, and i is 4; or n is 5, m is 4, and i is 3; or n is 6, m is 4 and i is 2; or n is 7, m is 4 and i is 1; n is 0, m is 5, and i is 7; or n is 1, m is 5, and i is 6; or n is 2, m is 5 and i is 5; or n is 3, m is 5, and i is 4; or n is 4, m is 5, and i is 3; or n is 5, m is 5, and i is 2; or n is 6, m is 5 and i is 1; n is 0, m is 6, and i is 6; or n is 1, m is 6, and i is 5; or n is 2, m is 6 and i is 4; or n is 3, m is 6, and i is 3; or n is 4, m is 6, and i is 2; or n is 5, m is 6, and i is 1; n is 0, m is 7, and i is 5; or n is 1, m is 7, and i is 4; or n is 2, m is 7 and i is 3; or n is 3, m is 7, and i is 2; or n is 4, m is 7, and i is 1; n is 0, m is 8, and i is 4; or n is 1, m is 8, and i is 3; or n is 2, m is 8 and i is 2; or n is 3, m is 8, and i is 1; n is 0, m is 9, and i is 3; or n is 1, m is 9, and i is 2; or n is 2, m is 9 and i is 1; n is 0, m is 10, and i is 2; or n is 1, m is 10, and i is 1; or n is 0, m is 11 and i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 12; or n is 1, m is 1, and i is 11; or n is 2, m is 1, and i is 10; or n is 3, m is 1, and i is 9; or n is 4, m is 1, and i is 8; or n is 5, m is 1, and i is 7; or n is 6, m is 1, and i is 6; or n is 7, m is 1, and i is 5; or n is 8, m is 1, and i is 4; or n is 9, m is 1, and i is 3; or n is 10, m is 1, and i is 2; or n is 11, m is 1 and i is 1; n is 0, m is 2, and i is 11; or n is 1, m is 2, and i is 10; or n is 2, m is 2, and i is 9; or n is 3, m is 2, and i is 8; or n is 4, m is 2, and i is 7; or n is 5, m is 2, and i is 6; or n is 6, m is 2, and i is 5; or n is 7, m is 2, and i is 4; or n is 8, m is 2, and i is 3; or n is 9, m is 2, and i is 2; or n is 10, m is 2, and i is 1; n is 0, m is 2, and i is 11; or n is 1, m is 2, and i is 10; or n is 2, m is 2, and i is 9; or n is 3, m is 2, and i is 8; or n is 4, m is 2, and i is 7; or n is 5, m is 2, and i is 6; or n is 6, m is 2, and i is 5; or n is 7, m is 2, and i is 4; or n is 8, m is 2, and i is 3; or n is 9, m is 2, and i is 2; or n is 10, m is 2, and i is 1; n is 0, m is 3, and i is 10; or n is 1, m is 3, and i is 9; or n is 2, m is 3, and i is 8; or n is 3, m is 3, and i is 7; or n is 4, m is 3, and i is 6, or n is 5, m is 3, and i is 5; or n is 6, m is 3, and i is 4; or n is 7, m is 3, and i is 3; or n is 8, m is 3, and i is 2; or n is 9, m is 3, and i is 1; n is 0, m is 4, and i is 9; or n is 1, m is 4, and i is 8; or n is 2, m is 4, and i is 7; or n is 3, m is 4, and i is 6; or n is 4, m is 4, and i is 5, or n is 5, m is 4, and i is 4; or n is 6, m is 4, and i is 3; or n is 7, m is 4, and i is 2; or n is 8, m is 4, and i is 1; n is 0, m is 5, and i is 8; or n is 1, m is 5, and i is 7; or n is 2, m is 5, and i is 6; or n is 3, m is 5, and i is 7; or n is 4, m is 5, and i is 4, or n is 5, m is 5, and i is 3; or n is 6, m is 5, and i is 2; or n is 7, m is 5, and i is 1; n is 0, m is 6, and i is 7; or n is 1, m is 6, and i is 6; or n is 2, m is 6, and i is 5; or n is 3, m is 6, and i is 4; or n is 4, m is 6, and i is 3, or n is 5, m is 6, and i is 2; or n is 6, m is 6, and i is 1; n is 0, m is 7, and i is 6; or n is 1, m is 7, and i is 5; or n is 2, m is 7, and i is 4; or n is 3, m is 7, and i is 3; or n is 4, m is 7, and i is 2, or n is 5, m is 7, and i is 1; n is 0, m is 8, and i is 5; or n is 1, m is 8, and i is 4; or n is 2, m is 8, and i is 3; or n is 3, m is 8, and i is 2; or n is 4, m is 8, and i is 1; n is 0, m is 9, and i is 4; or n is 1, m is 9, and i is 3; or n is 2, m is 9, and i is 2; or n is 3, m is 8, and i is 1; n is 0, m is 10, and i is 3; or n is 1, m is 10, and i is 2; or n is 2, m is 10, and i is 1; n is 0, m is 11, and i is 2; or n is 1, m is 11, and i is 1; or n is 0, m is 12, and i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 13; or n is 1, m is 1, and i is 12; or n is 2, m is 1, and i is 11; or n is 3, m is 1, and i is 10; or n is 4, m is 1, and i is 9; or n is 5, m is 1, and i is 8; or n is 6, m is 1, and i is 7; or n is 8, m is 1, and i is 5; or n is 9, m is 1, and i is 4; or n is 10, m is 1, and i is 3; or n is 11, m is 1, and i is 2; or n is 12, m is 1, and i is 1; n is 0, m is 2, and i is 12; or n is 1, m is 2, and i is 11; or n is 2, m is 2, and i is 10; or n is 3, m is 2, and i is 9; or n is 4, m is 2, and i is 8; or n is 5, m is 2, and i is 7; or n is 6, m is 2, and i is 6; or n is 7, m is 2, and i is 5, n is 8, m is 2, and i is 4; or n is 9, m is 2, and i is 3; or n is 10, m is 2, and i is 2; or n is 11, m is 2, and i is 1; n is 0, m is 3, and i is 11; or n is 1, m is 3, and i is 10; or n is 2, m is 3, and i is 9; or n is 3, m is 3, and i is 8; or n is 4, m is 3, and i is 7; or n is 5, m is 3, and i is 6; or n is 6, m is 3, and i is 5; or n is 7, m is 3, and i is 4; or n is 8, m is 3, and i is 3; or n is 9, m is 3, and i is 2; or n is 10, m is 3, and i is 1; n is 0, m is 4, and i is 10; or n is 1, m is 4, and i is 9; or n is 2, m is 4, and i is 8; or n is 3, m is 4, and i is 7; or n is 4, m is 4, and i is 6; or n is 5, m is 4, and i is 5; or n is 6, m is 4, and i is 4; or n is 7, m is 4, and i is 3; or n is 8, m is 4, and i is 2; or n is 9, m is 4, and i is 1; n is 0, m is 5, and i is 9; or n is 1, m is 5, and i is 8; or n is 2, m is 5, and i is 7; or n is 3, m is 5, and i is 6; or n is 4, m is 5, and i is 5; or n is 5, m is 5, and i is 4; or n is 6, m is 5, and i is 3; or n is 7, m is 5, and i is 2; or n is 8, m is 5, and i is 1; n is 0, m is 6, and i is 8; or n is 1, m is 6, and i is 7; or n is 2, m is 6, and i is 6; or n is 3, m is 6, and i is 5; or n is 4, m is 6, and i is 4; or n is 5, m is 6, and i is 3; or n is 6, m is 6, and i is 2; or n is 7, m is 6, and i is 1; n is 0, m is 7, and i is 7; or n is 1, m is 7, and i is 6; or n is 2, m is 7, and i is 5; or n is 3, m is 7, and i is 4; or n is 4, m is 7, and i is 3; or n is 5, m is 7, and i is 2; or n is 6, m is 7, and i is 1; n is 0, m is 8, and i is 6; or n is 1, m is 8, and i is 5; or n is 2, m is 8, and i is 4; or n is 3, m is 8, and i is 3; or n is 4, m is 8, and i is 2; or n is 5, m is 8, and i is 1; n is 0, m is 9, and i is 5; or n is 1, m is 9, and i is 4; or n is 2, m is 9, and i is 3; or n is 3, m is 9, and i is 2; or n is 4, m is 9, and i is 1; n is 0, m is 10, and i is 4; or n is 1, m is 10, and i is 3; or n is 2, m is 10, and i is 2; or n is 3, m is 10, and i is 1; n is 0, m is 11, and i is 3; or n is 1, m is 11, and i is 2; or n is 2, m is 11, and i is 1; n is 0, m is 12, and i is 2; or n is 1, m is 12, and i is 1; or n is 0, m is 13, and i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 14; or n is 1, m is 1, and i is 13; or n is 2, m is 1, and i is 12; or n is 3, m is 1, or i is 11; or n is 4, m is 1, and i is 10; or n is 5, m is 1, and i is 9; or n is 6, m is 1, and i is 8; or n is 7, m is 1, and i is 7; or n is 8, m is 1, and i is 6; or n is 9, m is 1, and i is 5; or n is 10, m is 1, and i is 4; or n is 11, m is 1 and i is 3; or n is 12, m is 1, and i is 2; or n is 13 and m is 1, and i is 1; n is 0, m is 2, and i is 13; or n is 1, m is 2, and i is 12; or n is 2, m is 2, and i is 11; or n is 3, m is 2, or i is 10; or n is 4, m is 2, and i is 9; or n is 5, m is 2, and i is 8; or n is 6, m is 2, and i is 7; or n is 7, m is 2, and i is 6; or n is 8, m is 2, and i is 5; or n is 9, m is 2, and i is 4; or n is 10, m is 2, and i is 3; or n is 11, m is 2 and i is 2; n is 12, m is 2 and i is 1; or n is 0, m is 3, and i is 12; or n is 1, m is 3, and i is 11; or n is 2, m is 3, and i is 10; or n is 3, m is 3, or i is 9; or n is 4, m is 3, and i is 8; or n is 5, m is 3, and i is 7; or n is 6, m is 3, and i is 6; or n is 7, m is 3, and i is 5; or n is 8, m is 3, and i is 4; or n is 9, m is 3, and i is 3; or n is 10, m is 3, and i is 2; or n is 11, m is 3 and i is 1; n is 0, m is 4, and i is 11; or n is 1, m is 4, and i is 10; or n is 2, m is 4, and i is 9; or n is 3, m is 4, or i is 8; or n is 4, m is 4, and i is 7; or n is 5, m is 4, and i is 6; or n is 6, m is 4, and i is 5; or n is 7, m is 4, and i is 4; or n is 8, m is 4, and i is 3; or n is 9, m is 4, and i is 2; or n is 10, m is 4, and i is 1; n is 0, m is 5, and i is 10; or n is 1, m is 5, and i is 9; or n is 2, m is 5, and i is 8; or n is 3, m is 5, or i is 7; or n is 4, m is 5, and i is 6; or n is 5, m is 5, and i is 5; or n is 6, m is 5, and i is 4; or n is 7, m is 5, and i is 3; or n is 8, m is 5, and i is 2; or n is 9, m is 5, and i is 1; n is 0, m is 6, and i is 9; or n is 1, m is 6, and i is 8; or n is 2, m is 6, and i is 7; or n is 3, m is 6, or i is 6; or n is 4, m is 6, and i is 5; or n is 5, m is 6, and i is 4; or n is 6, m is 6, and i is 3; or n is 7, m is 6, and i is 2; or n is 8, m is 6, and i is 1; n is 0, m is 7, and i is 8; or n is 1, m is 7, and i is 7; or n is 2, m is 7, and i is 6; or n is 3, m is 7, or i is 5; or n is 4, m is 7, and i is 4; or n is 5, m is 7, and i is 3; or n is 6, m is 7, and i is 2; or n is 7, m is 7, and i is 1; n is 0, m is 8, and i is 7; or n is 1, m is 8, and i is 6; or n is 2, m is 8, and i is 5; or n is 3, m is 8, or i is 4; or n is 4, m is 8, and i is 3; or n is 5, m is 8, and i is 2; or n is 6, m is 8, and i is 1; n is 0, m is 9, and i is 6; or n is 1, m is 9, and i is 5; or n is 2, m is 9, and i is 4; or n is 3, m is 9, or i is 3; or n is 4, m is 9, and i is 2; or n is 5, m is 9, and i is 1; n is 0, m is 10, and i is 5; or n is 1, m is 10, and i is 4; or n is 2, m is 10, and i is 3; or n is 3, m is 10, or i is 2; or n is 4, m is 10, and i is 1; n is 0, m is 11, and i is 4; or n is 1, m is 11, and i is 3; or n is 2, m is 11, and i is 2; or n is 3, m is 11, or i is 1; n is 0, m is 12, and i is 3; or n is 1, m is 12, and i is 2; or n is 2, m is 12, and i is 1; n is 0, m is 13, and i is 2; or n is 1, m is 13, and i is 1; n is 0, m is 14, and i is 1;

In some embodiments, for example, n is 0, m is 1, and i is 15; or n is 1, m is 1, and i is 14; or n is 2, m is 1 and i is 13; or n is 3, m is 1, and i is 12; or n is 4, m is 1, and i is 11; or n is 5, m is 1, and i is 10; or n is 6, m is 1 and i is 9; or n is 7, m is 1, and i is 8; or n is 9, m is 1, and i is 6; or n is 10, m is 1, and i is 5; or n is 11, m is 1, and i is 4; or n is 12, m is 1, and i is 3; or n is 13, m is 1, and i is 2; or n is 14, m is 1, and i is 1; n is 0, m is 2, and i is 14; or n is 1, m is 2, and i is 13; or n is 2, m is 2 and i is 12; or n is 3, m is 2, and i is 11; or n is 4, m is 2, and i is 10; or n is 5, m is 2, and i is 9; or n is 6, m is 2 and i is 8; or n is 7, m is 2, and i is 7; or n is 9, m is 2, and i is 5; or n is 10, m is 2, and i is 4; or n is 11, m is 2, and i is 3; or n is 12, m is 2, and i is 2; or n is 13, m is 1, and i is 1; n is 0, m is 3, and i is 13; or n is 1, m is 3, and i is 12; or n is 2, m is 3 and i is 11; or n is 3, m is 3, and i is 10; or n is 4, m is 3, and i is 9; or n is 5, m is 3, and i is 8; or n is 6, m is 3 and i is 7; or n is 7, m is 3, and i is 6; or n is 9, m is 3, and i is 4; or n is 10, m is 3, and i is 3; or n is 11, m is 3, and i is 2; or n is 12, m is 3, and i is 1; n is 0, m is 4, and i is 12; or n is 1, m is 4, and i is 11; or n is 2, m is 4 and i is 10; or n is 3, m is 4, and i is 9; or n is 4, m is 4, and i is 8; or n is 5, m is 4, and i is 7; or n is 6, m is 4 and i is 6; or n is 7, m is 4, and i is 5; or n is 8, m is 4, and i is 4; or n is 9, m is 4, and i is 3; or n is 10, m is 4, and i is 2; or n is 11, m is 4, and i is 1; n is 0, m is 5, and i is 11; or n is 1, m is 5, and i is 10; or n is 2, m is 5 and i is 9; or n is 3, m is 5, and i is 8; or n is 4, m is 5, and i is 7; or n is 5, m is 5, and i is 6; or n is 6, m is 5 and i is 5; or n is 7, m is 5, and i is 4; or n is 8, m is 5, and i is 3; or n is 9, m is 5, and i is 2; or n is 10, m is 5, and i is 1; n is 0, m is 6, and i is 10; or n is 1, m is 6, and i is 9; or n is 2, m is 6 and i is 8; or n is 3, m is 6, and i is 7; or n is 4, m is 6, and i is 6; or n is 5, m is 6, and i is 5; or n is 6, m is 6 and i is 4; or n is 7, m is 6, and i is 3; or n is 8, m is 6, and i is 2; or n is 9, m is 6, and i is 1; n is 0, m is 7, and i is 9; or n is 1, m is 7, and i is 8; or n is 2, m is 7 and i is 7; or n is 3, m is 7, and i is 6; or n is 4, m is 7, and i is 5; or n is 5, m is 7, and i is 4; or n is 6, m is 7 and i is 3; or n is 7, m is 7, and i is 2; or n is 8, m is 7, and i is 1; n is 0, m is 8, and i is 8; or n is 1, m is 8, and i is 7; or n is 2, m is 8 and i is 6; or n is 3, m is 8, and i is 5; or n is 4, m is 8, and i is 4; or n is 5, m is 8, and i is 3; or n is 6, m is 8 and i is 2; or n is 7, m is 8, and i is 1; n is 0, m is 9, and i is 7; or n is 1, m is 9, and i is 6; or n is 2, m is 9 and i is 5; or n is 3, m is 9, and i is 4; or n is 4, m is 9, and i is 3; or n is 5, m is 9, and i is 2; or n is 6, m is 9 and i is 1; n is 0, m is 10, and i is 6; or n is 1, m is 10, and i is 5; or n is 2, m is 10 and i is 4; or n is 3, m is 10, and i is 3; or n is 4, m is 10, and i is 2; or n is 5, m is 10, and i is 1; n is 0, m is 11, and i is 5; or n is 1, m is 11, and i is 4; or n is 2, m is 11 and i is 3; or n is 3, m is 11, and i is 2; or n is 4, m is 11, and i is 1; n is 0, m is 12, and i is 4; or n is 1, m is 12, and i is 3; or n is 2, m is 12 and i is 2; or n is 3, m is 12, and i is 1; n is 0, m is 13, and i is 3; or n is 1, m is 13, and i is 2; or n is 2, m is 13 and i is 1; n is 0, m is 14, and i is 2; or n is 1, m is 14, and i is 1; n is 0, m is 15, and i is 1; and positional isomers thereof.

In some embodiments, for example, n is 0, m is 1, and i is 16, or n is 1, m is 1, and i is 15; or n is 2, m is 1, and i is 14; or n is 3, m is 1, and i is 13; or n is 4, m is 1, and i is 12; or n is 5, m is 1 and i is 11; or n is 6, m is 1, and i is 10; or n is 7, m is 1, and i is 9; or n is 8, m is 1, and i is 8; or n is 9, m is 1, and i is 7; or n is 10, m is 1, and i is 6; or n is 11, m is 1, and i is 5; or n is 12, m is 1; and i is 4; or n is 13, m is 1, and i is 3; or n is 14, m is 1, and i is 2; or n is 15, m is 1 and i is 1; n is 0, m is 2, and i is 15, or n is 1, m is 2, and i is 14; or n is 2, m is 2, and i is 13; or n is 3, m is 2, and i is 12; or n is 4, m is 2, and i is 11; or n is 5, m is 2 and i is 10; or n is 6, m is 2, and i is 9; or n is 7, m is 2, and i is 8; or n is 8, m is 2, and i is 7; or n is 9, m is 2, and i is 6; or n is 10, m is 2, and i is 5; or n is 11, m is 2, and i is 4; or n is 12, m is 2; and i is 3; or n is 13, m is 2, and i is 2; or n is 14, m is 1, and i is 1; n is 0, m is 3, and i is 14, or n is 1, m is 3, and i is 13; or n is 2, m is 3, and i is 12; or n is 3, m is 3, and i is 11; or n is 4, m is 3, and i is 10; or n is 5, m is 3 and i is 9; or n is 6, m is 3, and i is 8; or n is 7, m is 3, and i is 7; or n is 8, m is 3, and i is 6; or n is 9, m is 3, and i is 5; or n is 10, m is 3, and i is 4; or n is 11, m is 3, and i is 3; or n is 12, m is 3; and i is 2; or n is 13, m is 3, and i is 1; n is 0, m is 4, and i is 13, or n is 1, m is 4, and i is 12; or n is 2, m is 4, and i is 11; or n is 3, m is 4, and i is 10; or n is 4, m is 4, and i is 9; or n is 5, m is 4 and i is 8; or n is 6, m is 4, and i is 7; or n is 7, m is 4, and i is 6; or n is 8, m is 4, and i is 5; or n is 9, m is 4, and i is 4; or n is 10, m is 4, and i is 3; or n is 11, m is 4, and i is 2; or n is 12, m is 4; and i is 1; n is 0, m is 5, and i is 12, or n is 1, m is 5, and i is 11; or n is 2, m is 5, and i is 10; or n is 3, m is 5, and i is 9; or n is 4, m is 5, and i is 8; or n is 5, m is 5 and i is 7; or n is 6, m is 5, and i is 6; or n is 7, m is 5, and i is 5; or n is 8, m is 5, and i is 4; or n is 9, m is 5, and i is 3; or n is 10, m is 5, and i is 2; or n is 11, m is 5, and i is 1; n is 0, m is 6, and i is 11, or n is 1, m is 6, and i is 10; or n is 2, m is 6, and i is 9; or n is 3, m is 6, and i is 8; or n is 4, m is 6, and i is 7; or n is 5, m is 6 and i is 6; or n is 6, m is 6, and i is 5; or n is 7, m is 6, and i is 4; or n is 8, m is 6, and i is 3; or n is 9, m is 6, and i is 2; or n is 10, m is 6, and i is 1; n is 0, m is 7, and i is 10, or n is 1, m is 7, and i is 9; or n is 2, m is 7, and i is 8; or n is 3, m is 7, and i is 7; or n is 4, m is 7, and i is 6; or n is 5, m is 7 and i is 5; or n is 6, m is 7, and i is 4; or n is 7, m is 7, and i is 3; or n is 8, m is 7, and i is 2; or n is 9, m is 7, and i is 1; n is 0, m is 8, and i is 9, or n is 1, m is 8, and i is 8; or n is 2, m is 8, and i is 7; or n is 3, m is 8, and i is 6; or n is 4, m is 8, and i is 5; or n is 5, m is 8 and i is 4; or n is 6, m is 8, and i is 3; or n is 7, m is 8, and i is 2; or n is 8, m is 8, and i is 1; n is 0, m is 9, and i is 8, or n is 1, m is 9, and i is 7; or n is 2, m is 9, and i is 6; or n is 3, m is 9, and i is 5; or n is 4, m is 9, and i is 4; or n is 5, m is 9 and i is 3; or n is 6, m is 9, and i is 2; or n is 7, m is 9, and i is 1; n is 0, m is 10, and i is 7, or n is 1, m is 10, and i is 6; or n is 2, m is 10, and i is 5; or n is 3, m is 10, and i is 4; or n is 4, m is 10, and i is 3; or n is 5, m is 10 and i is 2; or n is 6, m is 10, and i is 1; n is 0, m is 11, and i is 6, or n is 1, m is 11, and i is 5; or n is 2, m is 11, and i is 4; or n is 3, m is 11, and i is 3; or n is 4, m is 11, and i is 2; or n is 5, m is 11 and i is 1; n is 0, m is 12, and i is 5, or n is 1, m is 12, and i is 4; or n is 2, m is 12, and i is 3; or n is 3, m is 12, and i is 2; or n is 4, m is 12, and i is 1; n is 0, m is 13, and i is 4, or n is 1, m is 13, and i is 3; or n is 2, m is 13, and i is 2; or n is 3, m is 13, and i is 1; n is 0, m is 14, and i is 3, or n is 1, m is 14, and i is 2; or n is 2, m is 14, and i is 1; n is 0, m is 15, and i is 2, or n is 1, m is 15, and i is 1; or n is 0, m is 16, and i is 1; or positional isomers thereof.

Accordingly, some embodiments of the disclosure provide methods for preparing an olefinic alcohol product as described above, wherein the olefinic substrate is a metathesis product, and wherein the method includes: a) cross-metathesizing a first terminal olefin and a second different terminal olefin in the presence of a metathesis catalyst to form the metathesis product; and b) incubating the metathesis product with an enzyme capable of hydroxylating an subterminal carbon of the metathesis product to form an olefinic alcohol product.

In some embodiments, the first terminal olefin has the formula (CH₂═CH)(CH₂)_(m)H, the second different terminal olefin has the formula (CH₂═CH)(CH₂)_(n)H, the metathesis product has the formula H(CH₂)_(m)(CH═CH)(CH₂)_(n)H, the olefinic alcohol product has the formula H(CH₂)_(i)CHOH(CH₂)_(m-i-1)(CH═CH)(CH₂)_(n)H or H(CH₂)_(m)(CH═CH)(CH₂)_(n-i-1)CHOH(CH₂)_(i)H, and m, n and i are different integers between 1 and 17. In some embodiments, m, n, and i are different integers between 1 and 9.

The methods of the disclosure can also be conducted such that the biohydroxylation step is conducted prior to the metathesis step and/or other synthetic transformation steps. Accordingly, some embodiments of the disclosure provide methods wherein the olefinic substrate is a first terminal olefin, and wherein the method includes: a) incubating the first terminal olefin with an enzyme capable of hydroxylating an subterminal carbon of the terminal olefin to form an alkenol; and b) metathesizing the alkenol and a second terminal olefin in the presence of a metathesis catalyst to form the olefinic alcohol product.

The alcohol can be protected with a suitable protecting group if necessary. In some embodiments, the methods of the disclosure include: a) incubating the first terminal olefin with an enzyme capable of selectively hydroxylating an subterminal carbon of the terminal olefin to form an alkenol; b) protecting the alkenol to form a protected alkenol; c) metathesizing the protected alkenol and a second terminal olefin in the presence of a metathesis catalyst to form a protected olefinic alcohol product; and d) deprotecting the protected olefinic alcohol product to form the olefinic alcohol product.

Any suitable alcohol protecting group can be used in the methods of the disclosure. Such protecting groups are well known to one of ordinary skill in the art, including those that are disclosed in Protective Groups in Organic Synthesis, 4th edition, T. W. Greene and P. G. M. Wuts, John Wiley & Sons, New York, 2006, which is incorporated herein by reference in its entirety. In some embodiments, the α,ω-alkenol is protected via esterification and the should alkenol is protected via esterification with an acid selected from the group consisting of formate and acetate.

Synthesis of Terminal Alkenals

As indicated above, the alcohol moiety generated via hydroxylation can be further modified to generate alkenals or acetate esters.

Oxidation of Fatty Alcohols

Oxidation of fatty alcohols is often achieved via selective oxidation via pyridinium chlorochromate (PCC) (Scheme 9).

Alternatively, TEMPO (TEMPO=2,2,6,6-tetramethylpiperidinyl-N-oxyl) and related catalyst systems can be used to selectively oxidize alcohols to aldehydes. These methods are described in Ryland and Stahl (2014), herein incorporated by reference in its entirety.

Bio-Oxidation of Terminal Alcohols

Many insect pheromones are fatty aldehydes or comprise a fatty aldehyde component. As such, the conversion of the fatty alcohol produced via terminal hydroxylation to the fatty aldehyde is required to produce certain pheromones. The conversion of a fatty alcohol to a fatty aldehyde is known to be catalyzed by alcohol dehydrogenases (ADH) and alcohol oxidases (AOX). Additionally, the conversion of a length C_(n) fatty acid to a C_(n-1) fatty aldehyde is catalyzed by plant α-dioxygenases (α-DOX) (Scheme 10).

The present disclosure describes enzymes that oxidize fatty alcohols to fatty aldehydes.

In some embodiments, an alcohol oxidase (AOX) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. Alcohol oxidases catalyze the conversion of alcohols into corresponding aldehydes (or ketones) with electron transfer via the use of molecular oxygen to form hydrogen peroxide as a by-product. AOX enzymes utilize flavin adenine dinucleotide (FAD) as an essential cofactor and regenerate with the help of oxygen in the reaction medium. Catalase enzymes may be coupled with the AOX to avoid accumulation of the hydrogen peroxide via catalytic conversion into water and oxygen.

Based on the substrate specificities, AOXs may be categorized into four groups: (a) short chain alcohol oxidase, (b) long chain alcohol oxidase, (c) aromatic alcohol oxidase, and (d) secondary alcohol oxidase (Goswami et al. 2013). Depending on the chain length of the desired substrate, some member of these four groups are better suited than others as candidates for evaluation.

Short chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.13, Table 7) catalyze the oxidation of lower chain length alcohol substrates in the range of C1-C8 carbons (van der Klei et al. 1991) (Ozimek et al. 2005). Aliphatic alcohol oxidases from methylotrophic yeasts such as Candida boidinii and Komagataella pastoris (formerly Pichia pastoris) catalyze the oxidation of primary alkanols to the corresponding aldehydes with a preference for unbranched short-chain aliphatic alcohols. The most broad substrate specificity is found for alcohol oxidase from the Pichia pastoris including propargyl alcohol, 2-chloroethanol, 2-cyanoethanol (Dienys et al. 2003). The major challenge encountered in alcohol oxidation is the high reactivity of the aldehyde product. Utilization of a two liquid phase system (water/solvent) can provide in-situ removal of the aldehyde product from the reaction phase before it is further converted to the acid. For example, hexanal production from hexanol using Pichia pastoris alcohol oxidase coupled with bovine liver catalase was achieved in a bi-phasic system by taking advantage of the presence of a stable alcohol oxidase in aqueous phase (Karra-Chaabouni et al. 2003). For example, alcohol oxidase from Pichia pastoris was able to oxidize aliphatic alcohols of C6 to C11 when used biphasic organic reaction system (Murray and Duff 1990). Methods for using alcohol oxidases in a biphasic system according to (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) are incorporated by reference in their entirety.

Long chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.20; Table 8) include fatty alcohol oxidases, long chain fatty acid oxidases, and long chain fatty alcohol oxidases that oxidize alcohol substrates with carbon chain length of greater than six (Goswami et al. 2013). Banthorpe et al. reported a long chain alcohol oxidase purified from the leaves of Tanacetum vulgare that was able to oxidize saturated and unsaturated long chain alcohol substrates including hex-trans-2-en-1-ol and octan-1-ol (Banthorpe 1976) (Cardemil 1978). Other plant species, including Simmondsia chinensis (Moreau, R. A., Huang 1979), Arabidopsis thaliana (Cheng et al. 2004), and Lotus japonicas (Zhao et al. 2008) have also been reported as sources of long chain alcohol oxidases. Fatty alcohol oxidases are mostly reported from yeast species (Hommel and Ratledge 1990) (Vanhanen et al. 2000) (Hommel et al. 1994) (Kemp et al. 1990) and these enzymes play an important role in long chain fatty acid metabolism (Cheng et al. 2005). Fatty alcohol oxidases from yeast species that degrade and grow on long chain alkanes and fatty acid catalyze the oxidation of fatty alcohols. Fatty alcohol oxidase from Candida tropicalis has been isolated as microsomal cell fractions and characterized for a range of substrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991) (Mauersberger et al. 1992). Significant activity is observed for primary alcohols of length C₈ to C₁₆ with reported KM in the 10-50 μM range (Eirich et al. 2004). Alcohol oxidases described may be used for the conversion of medium chain aliphatic alcohols to aldehydes as described, for example, for whole-cells Candida boidinii (Gabelman and Luzio 1997), and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990). Long chain alcohol oxidases from filamentous fungi were produced during growth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha and Ratledge 1991). The long chain fatty alcohol oxidase (LjFAO1) from Lotus japonicas has been heterologously expressed in E. coli and exhibited broad substrate specificity for alcohol oxidation including 1-dodecanol and 1-hexadecanol (Zhao et al. 2008).

TABLE 7 Alcohol oxidase enzymes capable of oxidizing short chain alcohols (EC 1.1.3.13) Organism Gene names Accession No. Komagataella pastoris (strain ATCC 76273/ AOX1 PP7435_Chr4- F2QY27 CBS 7435/CECT 11047/NRRL Y-11430/ 0130 Wegner 21-1) (Yeast) (Pichia pastoris) Komagataella pastoris (strain GS115/ATCC AOX1 P04842 20864) (Yeast) (Pichia pastoris) PAS_chr4_0821 Komagataella pastoris (strain ATCC 76273/ AOX2 PP7435_Chr4- F2R038 CBS 7435/CECT 11047/NRRL Y-11430/ 0863 Wegner 21-1) (Yeast) (Pichia pastoris) Komagataella pastoris (strain GS115/ATCC AOX2 C4R702 20864) (Yeast) (Pichia pastoris) PAS_chr4_0152 Candida boidinii (Yeast) AOD1 Q00922 Pichia angusta (Yeast) (Hansenula polymorpha) MOX P04841 Thanatephorus cucumeris (strain AG1-IB/ AOD1 BN14_10802 M5CC52 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/ MOX BN14_12214 M5CF32 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/ AOD1 BN14_10691 M5CAV1 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/ AOD1 BN14_09479 M5C7F4 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/ AOD1 BN14_10803 M5CB66 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/ AOD1 BN14_09900 M5C9N9 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/ AOD1 BN14_08302 M5C2L8 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/ MOX BN14_09408 M5C784 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/ MOX BN14_09478 M5C8F8 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Thanatephorus cucumeris (strain AG1-IB/ AOD1 BN14_11356 M5CH40 isolate 7/3/14) (Lettuce bottom rot fungus) (Rhizoctonia solani) Ogataea henricii AOD1 A5LGF0 Candida methanosorbosa AOD1 A5LGE5 Candida methanolovescens AOD1 A5LGE4 Candida succiphila AOD1 A5LGE6 Aspergillus niger (strain CBS 513.88/FGSC An15g02200 A2R501 A1513) Aspergillus niger (strain CBS 513.88/FGSC An18g05480 A2RB46 A1513) Moniliophthora perniciosa (Witches'-broom I7CMK2 disease fungus) (Marasmius perniciosus) Candida cariosilignicola AOD1 A5LGE3 Candida pignaliae AOD1 A5LGE1 Candida pignaliae AOD2 A5LGE2 Candida sonorensis AOD1 A5LGD9 Candida sonorensis AOD2 A5LGE0 Pichia naganishii AOD1 A5LGF2 Ogataea minuta AOD1 A5LGF1 Ogataea philodendri AOD1 A5LGF3 Ogataea wickerhamii AOD1 A5LGE8 Kuraishia capsulata AOD1 A5LGE7 Talaromyces stipitatus (strain ATCC 10500/ TSTA_021940 B8MHF8 CBS 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Talaromyces stipitatus (strain ATCC 10500/ TSTA_065150 B8LTH7 CBS 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Talaromyces stipitatus (strain ATCC 10500/ TSTA_065150 B8LTH8 CBS 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Talaromyces stipitatus (strain ATCC 10500/ TSTA_000410 B8MSB1 CBS 375.48/QM 6759/NRRL 1006) (Penicillium stipitatum) Ogataea glucozyma AOD1 A5LGE9 Ogataea parapolymorpha (strain DL-1/ATCC HPODL_03886 W1QCJ3 26012/NRRL Y-7560) (Yeast) (Hansenula polymorpha) Gloeophyllum trabeum (Brown rot fungus) AOX A8DPS4 Pichia angusta (Yeast) (Hansenula polymorpha) mox1 A6PZG8 Pichia trehalophila AOD1 A5LGF4 Pichia angusta (Yeast) (Hansenula polymorpha) mox1 A6PZG9 Pichia angusta (Yeast) (Hansenula polymorpha) mox1 A6PZG7 Ixodes scapularis (Black-legged tick) (Deer IscW_ISCW017898 B7PIZ7 tick)

TABLE 8 Alcohol oxidase enzymes capable of oxidizing long chain alcohols including fatty alcohols (EC 1.1.3.20) Organism Gene names Accession No. Lotus japonicus (Lotus corniculatus var. FAO1 B5WWZ8 japonicus) Arabidopsis thaliana (Mouse-ear cress) FAO1 At1g03990 F21M11.7 Q9ZWB9 Lotus japonicus (Lotus corniculatus var. FAO2 B5WWZ9 japonicus) Arabidopsis thaliana (Mouse-ear cress) FAO3 At3g23410 MLM24.14 Q9LW56 MLM24.23 Arabidopsis thaliana (Mouse-ear cress) FAO4A At4g19380 O65709 T5K18.160 Arabidopsis thaliana (Mouse-ear cress) FAO4B At4g28570 T5F17.20 Q94BP3 Microbotryum violaceum (strain p1A1 MVLG_06864 U5HIL4 Lamole) (Anther smut fungus) (Ustilago violacea) Ajellomyces dermatitidis ATCC 26199 BDFG_03507 T5BNQ0 Gibberella zeae (strain PH-1/ATCC FG06918.1 FGSG_06918 I1RS14 MYA-4620/FGSC 9075/NRRL 31084) (Wheat head blight fungus) (Fusarium graminearum) Pichia sorbitophila (strain ATCC MYA- Piso0_004410 G8Y5E1 4447/BCRC 22081/CBS 7064/ GNLVRS01_PISO0K16268g NBRC 10061/NRRL Y-12695) GNLVRS01_PISO0L16269g (Hybrid yeast) Emericella nidulans (strain FGSC A4/ AN0623.2 ANIA_00623 Q5BFQ7 ATCC 38163/CBS 112.46/NRRL 194/ M139) (Aspergillus nidulans) Pyrenophora tritici-repentis (strain Pt- PTRG_10154 B2WJW5 1C-BFP) (Wheat tan spot fungus) (Drechslera tritici-repentis) Paracoccidioides lutzii (strain ATCC PAAG_09117 C1HEC6 MYA-826/Pb01) (Paracoccidioides brasiliensis) Candida parapsilosis (strain CDC 317/ CPAR2_204420 G8BG15 ATCC MYA-4646) (Yeast) (Monilia parapsilosis) Pseudozyma brasiliensis (strain PSEUBRA_SCAF2g03010 V5GPS6 GHG001) (Yeast) Candida parapsilosis (strain CDC 317/ CPAR2_204430 G8BG16 ATCC MYA-4646) (Yeast) (Monilia parapsilosis) Sclerotinia borealis F-4157 SBOR_5750 W9CDE2 Sordaria macrospora (strain ATCC SMAC_06361 F7W6K4 MYA-333/DSM 997/K(L3346)/K- hell) Sordaria macrospora (strain ATCC SMAC_01933 F7VSA1 MYA-333/DSM 997/K(L3346)/K- hell) Meyerozyma guilliermondii (strain PGUG_03467 A5DJL6 ATCC 6260/CBS 566/DSM 6381/ JCM 1539/NBRC 10279/NRRL Y- 324) (Yeast) (Candida guilliermondii) Trichophyton rubrum CBS 202.88 H107_00669 A0A023ATC5 Arthrobotrys oligospora (strain ATCC AOL_s00097g516 G1XJI9 24927/CBS 115.81/DSM 1491) (Nematode-trapping fungus) (Didymozoophaga oligospora) Scheffersomyces stipitis (strain ATCC FAO1 PICST_90828 A3LYX9 58785/CBS 6054/NBRC 10063/ NRRL Y-11545) (Yeast) (Pichia stipitis) Scheffersomyces stipitis (strain ATCC FAO2 PICST_32359 A3LW61 58785/CBS 6054/NBRC 10063/ NRRL Y-11545) (Yeast) (Pichia stipitis) Aspergillus oryzae (strain 3.042) Ao3042_09114 I8TL25 (Yellow koji mold) Fusarium oxysporum (strain Fo5176) FOXB_17532 F9GFU8 (Fusarium vascular wilt) Rhizopus delemar (strain RA 99-880/ RO3G_08271 I1C536 ATCC MYA-4621/FGSC 9543/ NRRL 43880) (Mucormycosis agent) (Rhizopus arrhizus var. delemar) Rhizopus delemar (strain RA 99-880/ RO3G_00154 I1BGX0 ATCC MYA-4621/FGSC 9543/ NRRL 43880) (Mucormycosis agent) (Rhizopus arrhizus var. delemar) Fusarium oxysporum (strain Fo5176) FOXB_07532 F9FMA2 (Fusarium vascular wilt) Penicillium roqueforti PROQFM164_S02g001772 W6QPY1 Aspergillus clavatus (strain ATCC 1007/ ACLA_018400 A1CNB5 CBS 513.65/DSM 816/NCTC 3887/ NRRL 1) Arthroderma otae (strain ATCC MYA- MCYG_08732 C5G1B0 4605/CBS 113480) (Microsporum canis) Trichophyton tonsurans (strain CBS TESG_07214 F2S8I2 112818) (Scalp ringworm fungus) Colletotrichum higginsianum (strain IMI CH063_13441 H1VUE7 349063) (Crucifer anthracnose fungus) Ajellomyces capsulatus (strain H143) HCDG_07658 C6HN77 (Darling's disease fungus) (Histoplasma capsulatum) Trichophyton rubrum (strain ATCC TERG_08235 F2T096 MYA-4607/CBS 118892) (Athlete's foot fungus) Cochliobolus heterostrophus (strain C5/ COCHEDRAFT_1201414 M2UMT9 ATCC 48332/race O) (Southern corn leaf blight fungus) (Bipolaris maydis) Candida orthopsilosis (strain 90-125) CORT_0D04510 H8X643 (Yeast) Candida orthopsilosis (strain 90-125) CORT_0D04520 H8X644 (Yeast) Candida orthopsilosis (strain 90-125) CORT_0D04530 H8X645 (Yeast) Pseudozyma aphidis DSM 70725 PaG_03027 W3VP49 Coccidioides posadasii (strain C735) CPC735_000380 C5P005 (Valley fever fungus) Magnaporthe oryzae (strain P131) (Rice OOW_P131scaffold01214g15 L7IZ92 blast fungus) (Pyricularia oryzae) Neurospora tetrasperma (strain FGSC NEUTE1DRAFT_82541 F8MKD1 2508/ATCC MYA-4615/P0657) Hypocrea virens (strain Gv29-8/FGSC TRIVIDRAFT_54537 G9MMY7 10586) (Gliocladium virens) (Trichoderma virens) Hypocrea virens (strain Gv29-8/FGSC TRIVIDRAFT_53801 G9MT89 10586) (Gliocladium virens) (Trichoderma virens) Aspergillus niger (strain CBS 513.88/ An01g09620 A2Q9Z3 FGSC A1513) Verticillium dahliae (strain VdLs.17/ VDAG_05780 G2X6J8 ATCC MYA-4575/FGSC 10137) (Verticillium wilt) Ustilago maydis (strain 521/FGSC UM02023.1 Q4PCZ0 9021) (Corn smut fungus) Fusarium oxysporum f. sp. lycopersici FOWG_13006 W9LNI9 MN25 Fusarium oxysporum f. sp. lycopersici FOWG_02542 W9N9Z1 MN25 Candida tropicalis (Yeast) FAO1 Q6QIR6 Magnaporthe oryzae (strain 70-15/ MGG_11317 G4MVK1 ATCC MYA-4617/FGSC 8958) (Rice blast fungus) (Pyricularia oryzae) Candida tropicalis (Yeast) faot Q9P8D9 Candida tropicalis (Yeast) FAO2a Q6QIR5 Phaeosphaeria nodorum (strain SN15/ SNOG_02371 Q0V0U3 ATCC MYA-4574/FGSC 10173) (Glume blotch fungus) (Septoria nodorum) Candida tropicalis (Yeast) FAO2b Q6QIR4 Pestalotiopsis fici W106-1 PFICI_11209 W3WU04 Magnaporthe oryzae (strain Y34) (Rice OOU_Y34scaffold00240g57 L7IFT5 blast fungus) (Pyricularia oryzae) Pseudogymnoascus destructans (strain GMDG_01756 L8G0G6 ATCC MYA-4855/20631-21) (Bat white-nose syndrome fungus) (Geomyces destructans) Pseudogymnoascus destructans (strain GMDG_04950 L8GCY2 ATCC MYA-4855/20631-21) (Bat white-nose syndrome fungus) (Geomyces destructans) Mycosphaerella fijiensis (strain MYCFIDRAFT_52380 M2Z831 CIRAD86) (Black leaf streak disease fungus) (Pseudocercospora fijiensis) Bipolaris oryzae ATCC 44560 COCMIDRAFT_84580 W7A0I8 Cladophialophora psammophila CBS A1O5_08147 W9WTM9 110553 Fusarium oxysporum f. sp. melonis FOMG_05173 X0AEE6 26406 Fusarium oxysporum f. sp. melonis FOMG_17829 W9ZBB7 26406 Cyphellophora europaea CBS 101466 HMPREF1541_02174 W252S5 Aspergillus kawachii (strain NBRC AKAW_00147 G7X626 4308) (White koji mold) (Aspergillus awamori var. kawachi) Aspergillus terreus (strain NIH 2624/ ATEG_05086 Q0CMJ8 FGSC A1156) Coccidioides immitis (strain RS) (Valley CIMG_02987 J3KAI8 fever fungus) Ajellomyces dermatitidis (strain ER-3/ BDCG_04701 C5GLS5 ATCC MYA-2586) (Blastomyces dermatitidis) Fusarium oxysporum f. sp. cubense FOC1_g10013865 N4U732 (strain race 1) (Panama disease fungus) Rhodotorula glutinis (strain ATCC RTG_00643 G0SVU8 204091/IIP 30/MTCC 1151) (Yeast) Aspergillus niger (strain ATCC 1015/ ASPNIDRAFT_35778 G3XTM6 CBS 113.46/FGSC A1144/LSHB Ac4/ NCTC 3858a/NRRL 328/USDA 3528.7) Candida cloacae fao1 Q9P8D8 Candida cloacae fao2 Q9P8D7 Fusarium oxysporum f. sp. cubense FOC1_g10006358 N4TUH3 (strain race 1) (Panama disease fungus) Candida albicans (strain SC5314/ FAO1 CaO19.13562 Q59RS8 ATCC MYA-2876) (Yeast) orf19.13562 Candida albicans (strain SC5314/ FAO1 CaO19.6143 Q59RP0 ATCC MYA-2876) (Yeast) orf19.6143 Chaetomium thermophilum (strain DSM CTHT_0018560 G0S2U9 1495/CBS 144.50/IMI 039719) Mucor circinelloides f. circinelloides HMPREF1544_05296 S2JDN0 (strain 1006PhL) (Mucormycosis agent) (Calyptromyces circinelloides) Mucor circinelloides f. circinelloides HMPREF1544_05295 S2JYP5 (strain 1006PhL) (Mucormycosis agent) (Calyptromyces circinelloides) Mucor circinelloides f. circinelloides HMPREF1544_06348 S2JVK9 (strain 1006PhL) (Mucormycosis agent) (Calyptromyces circinelloides) Botryotinia fuckeliana (strain BcDW1) BcDW1_6807 M7UD26 (Noble rot fungus) (Botrytis cinerea) Podospora anserina (strain S/ATCC PODANS_5_13040 B2AFD8 MYA-4624/DSM 980/FGSC 10383) (Pleurage anserina) Neosartorya fumigata (strain ATCC AFUA_1G17110 Q4WR91 MYA-4609/Af293/CBS 101355/ FGSC A1100) (Aspergillus fumigatus) Fusarium oxysporum f. sp. vasinfectum FOTG_00686 X0MEE6 25433 Fusarium oxysporum f. sp. vasinfectum FOTG_12485 X0LE98 25433 Trichophyton interdigitale H6 H101_06625 A0A022U717 Beauveria bassiana (strain ARSEF 2860) BBA_04100 J4UNY3 (White muscardine disease fungus) (Tritirachium shiotae) Fusarium oxysporum f. sp. radicis- FOCG_00843 X0GQ62 lycopersici 26381 Fusarium oxysporum f. sp. radicis- FOCG_15170 X0F4T1 lycopersici 26381 Neurospora tetrasperma (strain FGSC NEUTE2DRAFT_88670 G4UNN6 2509/P0656) Pseudozyma hubeiensis (strain SY62) PHSY_000086 R9NVU1 (Yeast) Lodderomyces elongisporus (strain LELG_03289 A5E102 ATCC 11503/CBS 2605/JCM 1781/ NBRC 1676/NRRL YB-4239) (Yeast) (Saccharomyces elongisporus) Malassezia globosa (strain ATCC MYA- MGL_3855 A8QAY8 4612/CBS 7966) (Dandruff-associated fungus) Byssochlamys spectabilis (strain No. 5/ PVAR5_7014 V5GBL6 NBRC 109023) (Paecilomyces variotii) Ajellomyces capsulatus (strain H88) HCEG_03274 F0UF47 (Darling's disease fungus) (Histoplasma capsulatum) Trichosporon asahii var. asahii (strain A1Q1_03669 J6FBP4 ATCC 90039/CBS 2479/JCM 2466/ KCTC 7840/NCYC 2677/UAMH 7654) (Yeast) Penicillium oxalicum (strain 114-2/ PDE_00027 S7Z8U8 CGMCC 5302) (Penicillium decumbens) Fusarium oxysporum f. sp. conglutinans FOPG_02304 X0IBE3 race 2 54008 Fusarium oxysporum f. sp. conglutinans FOPG_13066 X0H540 race 2 54008 Fusarium oxysporum f. sp. raphani FOQG_00704 X0D1G8 54005 Fusarium oxysporum f. sp. raphani FOQG_10402 X0C482 54005 Metarhizium acridum (strain CQMa MAC_03115 E9DZR7 102) Arthroderma benhamiae (strain ATCC ARB_02250 D4B1C1 MYA-4681/CBS 112371) (Trichophyton mentagrophytes) Fusarium oxysporum f. sp. cubense FOIG_12161 X0JFI6 tropical race 4 54006 Fusarium oxysporum f. sp. cubense FOIG_12751 X0JDU5 tropical race 4 54006 Cochliobolus heterostrophus (strain C4/ COCC4DRAFT_52836 N4WZZ0 ATCC 48331/race T) (Southern corn leaf blight fungus) (Bipolaris maydis) Trichosporon asahii var. asahii (strain A1Q2_00631 K1VZW1 CBS 8904) (Yeast) Mycosphaerella graminicola (strain CBS MYCGRDRAFT_37086 F9X375 115943/IPO323) (Speckled leaf blotch fungus) (Septoria tritici) Botryotinia fuckeliana (strain T4) BofuT4_P072020.1 G2XQ18 (Noble rot fungus) (Botrytis cinerea) Metarhizium anisopliae (strain ARSEF MAA_05783 E9F0I4 23/ATCC MYA-3075) Cladophialophora carrionii CBS 160.54 G647_05801 V9DAR1 Coccidioides posadasii (strain RMSCC CPSG_09174 E9DH75 757/Silveira) (Valley fever fungus) Rhodosporidium toruloides (strain RHTO_06879 M7X159 NP11) (Yeast) (Rhodotorula gracilis) Puccinia graminis f. sp. tritici (strain PGTG_10521 E3KIL8 CRL 75-36-700-3/race SCCL) (Black stem rust fungus) Trichophyton rubrum CBS 288.86 H103_00624 A0A022WG28 Colletotrichum fioriniae PJ7 CFIO01_08202 A0A010RKZ4 Trichophyton rubrum CBS 289.86 H104_00611 A0A022XB46 Cladophialophora yegresii CBS 114405 A1O7_02579 W9WC55 Colletotrichum orbiculare (strain 104-T/ Cob_10151 N4VFP3 ATCC 96160/CBS 514.97/LARS 414/ MAFF 240422) (Cucumber anthracnose fungus) (Colletotrichum lagenarium) Drechslerella stenobrocha 248 DRE_03459 W7IDL6 Neosartorya fumigata (strain CEA10/ AFUB_016500 B0XP90 CBS 144.89/FGSC A1163) (Aspergillus fumigatus) Thielavia terrestris (strain ATCC 38088/ THITE_2117674 G2R8H9 NRRL 8126) (Acremonium alabamense) Gibberella fujikuroi (strain CBS 195.34/ FFUJ_02948 S0DZP7 IMI 58289/NRRL A-6831) (Bakanae and foot rot disease fungus) (Fusarium fujikuroi) Gibberella fujikuroi (strain CBS 195.34/ FFUJ_12030 S0EMC6 IMI 58289/NRRL A-6831) (Bakanae and foot rot disease fungus) (Fusarium fujikuroi) Aspergillus flavus (strain ATCC 200026/ AFLA_109870 B8N941 FGSC A1120/NRRL 3357/JCM 12722/SRRC 167) Togninia minima (strain UCR-PA7) UCRPA7_1719 R8BTZ6 (Esca disease fungus) (Phaeoacremonium aleophilum) Ajellomyces dermatitidis (strain ATCC BDDG_09783 F2TUC0 18188/CBS 674.68) (Blastomyces dermatitidis) Macrophomina phaseolina (strain MS6) MPH_10582 K2RHA5 (Charcoal rot fungus) Neurospora crassa (strain ATCC 24698/ NCU08977 Q7S2Z2 74-OR23-1A/CBS 708.71/DSM 1257/ FGSC 987) Neosartorya fischeri (strain ATCC 1020/ NFIA_008260 A1D156 DSM 3700/FGSC A1164/NRRL 181) (Aspergillus fischerianus) Fusarium pseudograminearum (strain FPSE_11742 K3U9J5 CS3096) (Wheat and barley crown-rot fungus) Spathaspora passalidarum (strain NRRL SPAPADRAFT_54193 G3AJP0 Y-27907/11-Y1) Spathaspora passalidarum (strain NRRL SPAPADRAFT_67198 G3ANX7 Y-27907/11-Y1) Trichophyton verrucosum (strain HKI TRV_07960 D4DL86 0517) Arthroderma gypseum (strain ATCC MGYG_07264 E4V2J0 MYA-4604/CBS 118893) (Microsporum gypseum) Hypocrea jecorina (strain QM6a) TRIREDRAFT_43893 G0R7P8 (Trichoderma reesei) Trichophyton rubrum MR1448 H110_00629 A0A022Z1G4 Aspergillus ruber CBS 135680 EURHEDRAFT_512125 A0A017SPR0 Glarea lozoyensis (strain ATCC 20868/ GLAREA_04397 S3D6C1 MF5171) Setosphaeria turcica (strain 28A) SETTUDRAFT_20639 R0K6H8 (Northern leaf blight fungus) (Exserohilum turcicum) Paracoccidioides brasiliensis (strain PADG_06552 C1GH16 Pb18) Fusarium oxysporum Fo47 FOZG_13577 W9JPG9 Fusarium oxysporum Fo47 FOZG_05344 W9KPH3 Trichophyton rubrum MR1459 H113_00628 A0A022ZY09 Penicillium marneffei (strain ATCC PMAA_075740 B6QBY3 18224/CBS 334.59/QM 7333) Sphaerulina musiva (strain SO2202) SEPMUDRAFT_154026 M3DAK6 (Poplar stem canker fungus) (Septoria musiva) Gibberella moniliformis (strain M3125/ FVEG_10526 W7N4P8 FGSC 7600) (Maize ear and stalk rot fungus) (Fusarium verticillioides) Gibberella moniliformis (strain M3125/ FVEG_08281 W7MVR9 FGSC 7600) (Maize ear and stalk rot fungus) (Fusarium verticillioides) Pseudozyma antarctica (strain T-34) PANT_22d00298 M9MGF2 (Yeast) (Candida antarctica) Paracoccidioides brasiliensis (strain PABG_07795 C0SJD4 Pb03) Rhizophagus irregularis (strain DAOM GLOINDRAFT_82554 U9TF61 181602/DAOM 197198/MUCL 43194) (Arbuscular mycorrhizal fungus) (Glomus intraradices) Penicillium chrysogenum (strain ATCC Pc21g23700 B6HJ58 28089/DSM 1075/Wisconsin 54- PCH_Pc21g23700 1255) (Penicillium notatum) Baudoinia compniacensis (strain UAMH BAUCODRAFT_274597 M2M6Z5 10762) (Angels' share fungus) Hypocrea atroviridis (strain ATCC TRIATDRAFT_280929 G9NJ32 20476/IMI 206040) (Trichoderma atroviride) Colletotrichum gloeosporioides (strain CGLO_06642 T0LPH0 Cg-14) (Anthracnose fungus) (Glomerella cingulata) Cordyceps militaris (strain CM01) CCM_02665 G3JB34 (Caterpillar fungus) Pyronema omphalodes (strain CBS PCON_13062 U4LKE9 100304) (Pyronema confluens) Colletotrichum graminicola (strain GLRG_08499 E3QR67 M1.001/M2/FGSC 10212) (Maize anthracnose fungus) (Glomerella graminicola) Glarea lozoyensis (strain ATCC 74030/ M7I_2117 H0EHX4 MF5533) Fusarium oxysporum f. sp. cubense FOC4_g10002493 N1S969 (strain race 4) (Panama disease fungus) Fusarium oxysporum f. sp. cubense FOC4_g10011461 N1RT80 (strain race 4) (Panama disease fungus) Cochliobolus sativus (strain ND90Pr/ COCSADRAFT_295770 M2TBE4 ATCC 201652) (Common root rot and spot blotch fungus) (Bipolaris sorokiniana) Mixia osmundae (strain CBS 9802/ Mo05571 E5Q_05571 G7E7S3 IAM 14324/JCM 22182/KY 12970) Mycosphaerella pini (strain NZE10/ DOTSEDRAFT_69651 N1PXR0 CBS 128990) (Red band needle blight fungus) (Dothistroma septosporum) Grosmannia clavigera (strain kw1407/ CMQ_1113 F0XC64 UAMH 11150) (Blue stain fungus) (Graphiocladiella clavigera) Fusarium oxysporum FOSC 3-a FOYG_03004 W9IUE5 Fusarium oxysporum FOSC 3-a FOYG_16040 W9HNP0 Fusarium oxysporum FOSC 3-a FOYG_17058 W9HB31 Nectria haematococca (strain 77-13-4/ NECHADRAFT_37686 C7YQL1 ATCC MYA-4622/FGSC 9596/ MPVI) (Fusarium solani subsp. pisi) Nectria haematococca (strain 77-13-4/ NECHADRAFT_77262 C7ZJI0 ATCC MYA-4622/FGSC 9596/ MPVI) (Fusarium solani subsp. pisi) Tuber melanosporum (strain Mel28) GSTUM_00010376001 D5GLS0 (Perigord black truffle) Ajellomyces dermatitidis (strain BDBG_07633 C5JYI9 SLH14081) (Blastomyces dermatitidis) Chaetomium globosum (strain ATCC CHGG_09885 Q2GQ69 6205/CBS 148.51/DSM 1962/NBRC 6347/NRRL 1970) (Soil fungus) Candida tenuis (strain ATCC 10573/ CANTEDRAFT_108652 G3B9Z1 BCRC 21748/CBS 615/JCM 9827/ NBRC 10315/NRRL Y-1498/VKM Y-70) (Yeast) Trichophyton rubrum CBS 100081 H102_00622 A0A022VKY4 Pyrenophora teres f. teres (strain 0-1) PTT_09421 E3RLZ3 (Barley net blotch fungus) (Drechslera teres f. teres) Colletotrichum gloeosporioides (strain CGGC5_4608 L2GB29 Nara gc5) (Anthracnose fungus) (Glomerella cingulata) Gibberella zeae (Wheat head blight FG05_06918 A0A016PCS4 fungus) (Fusarium graminearum) Trichophyton soudanense CBS 452.61 H105_00612 A0A022Y6A6 Sclerotinia sclerotiorum (strain ATCC SS1G_07437 A7EQ37 18683/1980/Ss-1) (White mold) (Whetzelinia sclerotiorum) Fusarium oxysporum f. sp. pisi HDV247 FOVG_14401 W9NWU8 Fusarium oxysporum f. sp. pisi HDV247 FOVG_02874 W9Q5V3 Ustilago hordei (strain Uh4875-4) UHOR_03009 I2G1Z4 (Barley covered smut fungus) Sporisorium reilianum (strain SRZ2) sr12985 E6ZYF7 (Maize head smut fungus) Bipolaris zeicola 26-R-13 COCCADRAFT_81154 W6YIP8 Melampsora larici-populina (strain MELLADRAFT_78490 F4RUZ8 98AG31/pathotype 3-4-7) (Poplar leaf rust fungus) Fusarium oxysporum f. sp. lycopersici FOXG_01901 J9MG95 (strain 4287/CBS 123668/FGSC 9935/ NRRL 34936) (Fusarium vascular wilt of tomato) Fusarium oxysporum f. sp. lycopersici FOXG_11941 J9N9S4 (strain 4287/CBS 123668/FGSC 9935/ NRRL 34936) (Fusarium vascular wilt of tomato) Bipolaris victoriae FI3 COCVIDRAFT_39053 W7EMJ8 Debaryomyces hansenii (strain ATCC DEHA2E04268g Q6BQL4 36239/CBS 767/JCM 1990/NBRC 0083/IGC 2968) (Yeast) (Torulaspora hansenii) Clavispora lusitaniae (strain ATCC CLUG_01505 C4XZX3 42720) (Yeast) (Candida lusitaniae) Candida albicans (strain WO-1) (Yeast) CAWG_02023 C4YME4 Trichophyton rubrum MR850 H100_00625 A0A022U0Q2 Candida dubliniensis (strain CD36/ CD36_32890 B9WMC7 ATCC MYA-646/CBS 7987/NCPF 3949/NRRL Y-17841) (Yeast) Starmerella bombicola AOX1 A0A024FB95 Thielavia heterothallica (strain ATCC MYCTH_103590 G2QJL7 42464/BCRC 31852/DSM 1799) (Myceliophthora thermophila) Claviceps purpurea (strain 20.1) (Ergot CPUR_07614 M1WFI4 fungus) (Sphacelia segetum) Aspergillus oryzae (strain ATCC 42149/ AO090023000571 Q2UH61 RIB 40) (Yellow koji mold) Dictyostelium discoideum (Slime mold) DDB_0184181 Q54DT6 DDB_G0292042 Triticum urartu (Red wild einkorn) TRIUR3_22733 M7YME5 (Crithodium urartu) Solanum tuberosum (Potato) PGSC0003DMG400017211 M1BG07 Oryza sativa subsp. japonica (Rice) OSJNBb0044B19.5 Q8W5P8 LOC_Os10g33540 Oryza sativa subsp. japonica (Rice) OJ1234_B11.20 Q6K9N5 Os02g0621800 Oryza sativa subsp. japonica (Rice) OSJNBa0001K12.5 Q8W5P3 LOC_Os10g33520 Zea mays (Maize) ZEAMMB73_809149 C0P3J6 Citrus clementina CICLE_v10011111mg V4S9P4 Citrus clementina CICLE_v10018992mg V4U4C9 Citrus clementina CICLE_v10004405mg V4S9D3 Citrus clementina CICLE_v10004403mg V4RZZ6 Morus notabilis L484_011703 W9RIK0 Morus notabilis L484_005930 W9RET7 Medicago truncatula (Barrel medic) MTR_1g075650 G7I4U3 (Medicago tribuloides) Arabidopsis thaliana (Mouse-ear cress) Q8LDP0 Medicago truncatula (Barrel medic) MTR_4g081080 G7JF07 (Medicago tribuloides) Simmondsia chinensis (Jojoba) (Buxus L7VFV2 chinensis) Prunus persica (Peach) (Amygdalus PRUPE_ppa018458mg M5VXL1 persica) Aphanomyces astaci H257_07411 W4GI89 Aphanomyces astaci H257_07412 W4GI44 Aphanomyces astaci H257_07411 W4GKE3 Aphanomyces astaci H257_07411 W4GK29 Aphanomyces astaci H257_07411 W4GJ79 Aphanomyces astaci H257_07411 W4GI38 Phaeodactylum tricornutum (strain PHATRDRAFT_48204 B7G6C1 CCAP 1055/1) Hordeum vulgare var. distichum (Two- F2E4R4 rowed barley) Hordeum vulgare var. distichum (Two- F2DZG1 rowed barley) Hordeum vulgare var. distichum (Two- M0YPG7 rowed barley) Hordeum vulgare var. distichum (Two- M0YPG6 rowed barley) Hordeum vulgare var. distichum (Two- F2CUY4 rowed barley) Ricinus communis (Castor bean) RCOM_0867830 B9S1S3 Brassica rapa subsp. pekinensis (Chinese BRA014947 M4DEM5 cabbage) (Brassica pekinensis) Ricinus communis (Castor bean) RCOM_0258730 B9SV13 Brassica rapa subsp. pekinensis (Chinese BRA001912 M4CCI2 cabbage) (Brassica pekinensis) Brassica rapa subsp. pekinensis (Chinese BRA012548 M4D7T8 cabbage) (Brassica pekinensis) Brassica rapa subsp. pekinensis (Chinese BRA024190 M4E5Y6 cabbage) (Brassica pekinensis) Brassica rapa subsp. pekinensis (Chinese BRA015283 M4DFL0 cabbage) (Brassica pekinensis) Ricinus communis (Castor bean) RCOM_1168730 B9SS54 Zea mays (Maize) C4J691 Oryza glaberrima (African rice) I1P2B7 Zea mays (Maize) B6SXM3 Zea mays (Maize) C0HFU4 Aegilops tauschii (Tausch's goatgrass) F775_19577 R7W4J3 (Aegilops squarrosa) Solanum habrochaites (Wild tomato) R9R6T0 (Lycopersicon hirsutum) Physcomitrella patens subsp. patens PHYPADRAFT_124285 A9S535 (Moss) Physcomitrella patens subsp. patens PHYPADRAFT_113581 A9RG13 (Moss) Physcomitrella patens subsp. patens PHYPADRAFT_182504 A9S9A5 (Moss) Solanum pennellii (Tomato) R9R6Q1 (Lycopersicon pennellii) Vitis vinifera (Grape) VIT_02s0087g00630 F6HJ27 Vitis vinifera (Grape) VIT_07s0005g03780 F6HZM3 Vitis vinifera (Grape) VIT_05s0049g01400 F6H8T4 Vitis vinifera (Grape) VITISV_019349 A5AH38 Capsella rubella CARUB_v10013046mg R0HIT3 Capsella rubella CARUB_v10004212mg R0GUX4 Capsella rubella CARUB_v10004208mg R0F3X6 Capsella rubella CARUB_v10012453mg R0ILD0 Capsella rubella CARUB_v10004208mg R0GUX1 Eutrema salsugineum (Saltwater cress) EUTSA_v10024496mg V4MD54 (Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress) EUTSA_v10020141mg V4NM59 (Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress) EUTSA_v10024496mg V4LUR9 (Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress) EUTSA_v10024528mg V4P767 (Sisymbrium salsugineum) Eutrema salsugineum (Saltwater cress) EUTSA_v10006882mg V4L2P6 (Sisymbrium salsugineum) Selaginella moellendorffii (Spikemoss) SELMODRAFT_87684 D8R6Z6 Selaginella moellendorffii (Spikemoss) SELMODRAFT_87621 D8R6Z5 Selaginella moellendorffii (Spikemoss) SELMODRAFT_74601 D8QN81 Selaginella moellendorffii (Spikemoss) SELMODRAFT_73531 D8QN82 Sorghum bicolor (Sorghum) (Sorghum Sb04g026390 C5XXS4 vulgare) SORBIDRAFT_04g026390 Sorghum bicolor (Sorghum) (Sorghum Sb04g026370 C5XXS1 vulgare) SORBIDRAFT_04g026370 Sorghum bicolor (Sorghum) (Sorghum Sb01g019470 C5WYH6 vulgare) SORBIDRAFT_01g019470 Sorghum bicolor (Sorghum) (Sorghum Sb01g019480 C5WYH7 vulgare) SORBIDRAFT_01g019480 Sorghum bicolor (Sorghum) (Sorghum Sb01g019460 C5WYH5 vulgare) SORBIDRAFT_01g019460 Solanum pimpinellifolium (Currant R9R6J2 tomato) (Lycopersicon pimpinellifolium) Phaseolus vulgaris (Kidney bean) PHAVU_007G124200g V7BGM7 (French bean) Phaseolus vulgaris (Kidney bean) PHAVU_011G136600g V7AI35 (French bean) Phaseolus vulgaris (Kidney bean) PHAVU_001G162800g V7D063 (French bean) Solanum tuberosum (Potato) PGSC0003DMG400024294 M1C923 Solanum tuberosum (Potato) PGSC0003DMG400018458 M1BKV4 Solanum tuberosum (Potato) PGSC0003DMG400018458 M1BKV3 Glycine max (Soybean) (Glycine K7LK61 hispida) Glycine max (Soybean) (Glycine K7KXQ9 hispida) Populus trichocarpa (Western balsam POPTR_0008s16920g B9HKS3 poplar) (Populus balsamifera subsp. trichocarpa) Picea sitchensis (Sitka spruce) (Pinus B8LQ84 sitchensis) Populus trichocarpa (Western balsam POPTR_0004s24310g U5GKQ5 poplar) (Populus balsamifera subsp. trichocarpa) Populus trichocarpa (Western balsam POPTR_0010s07980g B9HSG9 poplar) (Populus balsamifera subsp. trichocarpa) Glycine max (Soybean) (Glycine I1N9S7 hispida) Glycine max (Soybean) (Glycine I1LSK5 hispida) Setaria italica (Foxtail millet) (Panicum Si034362m.g K4A658 italicum) Solanum lycopersicum (Tomato) Solyc09g072610.2 K4CUT7 (Lycopersicon esculentum) Setaria italica (Foxtail millet) (Panicum Si016380m.g K3YQ38 italicum) Solanum lycopersicum (Tomato) R9R6I9 (Lycopersicon esculentum) Solanum lycopersicum (Tomato) Solyc09g090350.2 K4CW61 (Lycopersicon esculentum) Solanum lycopersicum (Tomato) Solyc08g005630.2 K4CI54 (Lycopersicon esculentum) Solanum lycopersicum (Tomato) Solyc08g075240.2 K4CMP1 (Lycopersicon esculentum) Setaria italica (Foxtail millet) (Panicum Si034359m.g K4A655 italicum) Setaria italica (Foxtail millet) (Panicum Si034354m.g K4A650 italicum) Mimulus guttatus (Spotted monkey MIMGU_mgv1a001896mg A0A022PU07 flower) (Yellow monkey flower) Mimulus guttatus (Spotted monkey MIMGU_mgv1a022390mg A0A022RAV4 flower) (Yellow monkey flower) Mimulus guttatus (Spotted monkey MIMGU_mgv1a001868mg A0A022S2E6 flower) (Yellow monkey flower) Mimulus guttatus (Spotted monkey MIMGU_mgv1a001883mg A0A022S275 flower) (Yellow monkey flower) Mimulus guttatus (Spotted monkey MIMGU_mgv1a001761mg A0A022QNF0 flower) (Yellow monkey flower) Musa acuminata subsp. malaccensis M0SNA8 (Wild banana) (Musa malaccensis) Musa acuminata subsp. malaccensis M0RUT7 (Wild banana) (Musa malaccensis) Musa acuminata subsp. malaccensis M0RUK3 (Wild banana) (Musa malaccensis) Saprolegnia diclina VS20 SDRG_1901 T0RG89 Brachypodium distachyon (Purple false BRADI3G49085 I1IBP7 brome) (Trachynia distachya) Brachypodium distachyon (Purple false BRADI3G28677 I1I4N2 brome) (Trachynia distachya) Brachypodium distachyon (Purple false BRADI3G28657 I1I4N0 brome) (Trachynia distachya) Oryza sativa subsp. indica (Rice) OsI_34012 B8BHG0 Oryza sativa subsp. indica (Rice) OsI_08118 B8AFT8 Oryza sativa subsp. indica (Rice) OsI_34008 A2Z8H1 Oryza sativa subsp. indica (Rice) OsI_34014 B8BHG1 Oryza sativa subsp. japonica (Rice) LOC_Os10g33460 Q7XDG3 Oryza sativa subsp. japonica (Rice) Os10g0474800 Q0IX12 Oryza sativa subsp. japonica (Rice) Os10g0474966 C7J7R1 Oryza sativa subsp. japonica (Rice) OSJNBa0001K12.13 Q8W5N7 Oryza sativa subsp. japonica (Rice) OsJ_31873 B9G683 Oryza sativa subsp. japonica (Rice) OsJ_31875 B9G684 Oryza sativa subsp. japonica (Rice) OSJNBa0001K12.3 Q8W5P5 Arabidopsis lyrata subsp. lyrata (Lyre- ARALYDRAFT_470376 D7KDA3 leaved rock-cress) Arabidopsis lyrata subsp. lyrata (Lyre- ARALYDRAFT_479855 D7L3B6 leaved rock-cress) Arabidopsis lyrata subsp. lyrata (Lyre- ARALYDRAFT_491906 D7MDA9 leaved rock-cress) Arabidopsis lyrata subsp. lyrata (Lyre- ARALYDRAFT_914728 D7MGS9 leaved rock-cress)

In some embodiments, an alcohol dehydrogenase (ADH, Table 9) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. A number of ADHs identified from alkanotrophic organisms, Pseudomonas fluorescens NRRL B-1244 (Hou et al. 1983), Pseudomonas butanovora ATCC 43655 (Vangnai and Arp 2001), and Acinetobacter sp. strain M-1 (Tani et al. 2000), have shown to be active on short to medium-chain alkyl alcohols (C₂ to C₁₄). Additionally, commercially available ADHs from Sigma, Horse liver ADH and Baker's yeast ADH have detectable activity for substrates with length C₁₀ and greater. The reported activities for the longer fatty alcohols may be impacted by the difficulties in solubilizing the substrates. For the yeast ADH from Sigma, little to no activity is observed for C₁₂ to C₁₄ aldehydes by (Tani et al. 2000), however, activity for C₁₂ and C₁₆ hydroxy-w-fatty acids has been observed (Lu et al. 2010). Recently, two ADHs were characterized from Geobacillus thermodenitrificans NG80-2, an organism that degrades Cis to C₃₆ alkanes using the LadA hydroxylase. Activity was detected from methanol to 1-triacontanol (C₃₀) for both ADHs, with 1-octanol being the preferred substrate for ADH2 and ethanol for ADH1 (Liu et al. 2009).

The use of ADHs in whole-cell bioconversions has been mostly focused on the production of chiral alcohols from ketones (Ernst et al. 2005) (Schroer et al. 2007). Using the ADH from Lactobacillus brevis and coupled cofactor regeneration with isopropanol, Schroer et al. reported the production of 797 g of (R)-methyl-3 hydroxybutanoate from methyl acetoacetate, with a space time yield of 29 g/L/h (Schroer et al. 2007). Examples of aliphatic alcohol oxidation in whole-cell transformations have been reported with commercially obtained S. cerevisiae for the conversion of hexanol to hexanal (Presecki et al. 2012) and 2-heptanol to 2-heptanone (Cappaert and Larroche 2004).

TABLE 9 Exemplary alcohol dehydrogenase enzymes. Organisms Gene Name Accession No. Bactrocera oleae (Olive fruit fly) (Dacus oleae) ADH Q9NAR7 Cupriavidus necator (Alcaligenes eutrophus) adh P14940 (Ralstonia eutropha) Drosophila adiastola (Fruit fly) (Idiomyia Adh Q00669 adiastola) Drosophila affinidisjuncta (Fruit fly) (Idiomyia Adh P21518 affinidisjuncta) Drosophila ambigua (Fruit fly) Adh P25139 Drosophila borealis (Fruit fly) Adh P48584 Drosophila differens (Fruit fly) Adh P22245 Drosophila equinoxialis (Fruit fly) Adh Q9NG42 Drosophila flavomontana (Fruit fly) Adh P48585 Drosophila guanche (Fruit fly) Adh Q09009 Drosophila hawaiiensis (Fruit fly) Adh P51549 Drosophila heteroneura (Fruit fly) Adh P21898 Drosophila immigrans (Fruit fly) Adh Q07588 Drosophila insularis (Fruit fly) Adh Q9NG40 Drosophila lebanonensis (Fruit fly) Adh P10807 (Scaptodrosophila lebanonensis) Drosophila mauritiana (Fruit fly) Adh P07162 Drosophila madeirensis (Fruit fly) Adh Q09010 Drosophila mimica (Fruit fly) (Idiomyia mimica) Adh Q00671 Drosophila nigra (Fruit fly) (Idiomyia nigra) Adh Q00672 Drosophila orena (Fruit fly) Adh P07159 Drosophila pseudoobscura bogotana (Fruit fly) Adh P84328 Drosophila picticornis (Fruit fly) (Idiomyia Adh P23361 picticornis) Drosophila planitibia (Fruit fly) Adh P23277 Drosophila paulistorum (Fruit fly) Adh Q9U8S9 Drosophila silvestris (Fruit fly) Adh P23278 Drosophila subobscura (Fruit fly) Adh Q03384 Drosophila teissieri (Fruit fly) Adh P28484 Drosophila tsacasi (Fruit fly) Adh P51550 Fragaria ananassa (Strawberry) ADH P17648 Malus domestica (Apple) (Pyrus malus) ADH P48977 Scaptomyza albovittata (Fruit fly) Adh P25988 Scaptomyza crassifemur (Fruit fly) (Drosophila Adh Q00670 crassifemur) Sulfolobus sp. (strain RC3) adh P50381 Zaprionus tuberculatus (Vinegar fly) Adh P51552 Geobacillus stearothermophilus (Bacillus adh P42327 stearothermophilus) Drosophila mayaguana (Fruit fly) Adh, Adh2 P25721 Drosophila melanogaster (Fruit fly) Adh, CG3481 P00334 Drosophila pseudoobscura pseudoobscura (Fruit Adh, GA17214 Q6LCE4 fly) Drosophila simulans (Fruit fly) Adh, GD23968 Q24641 Drosophila yakuba (Fruit fly) Adh, GE19037 P26719 Drosophila ananassae (Fruit fly) Adh, GF14888 Q50L96 Drosophila erecta (Fruit fly) Adh, GG25120 P28483 Drosophila grimshawi (Fruit fly) (Idiomyia Adh, GH13025 P51551 grimshawi) Drosophila willistoni (Fruit fly) Adh, GK18290 Q05114 Drosophila persimilis (Fruit fly) Adh, GL25993 P37473 Drosophila sechellia (Fruit fly) Adh, GM15656 Q9GN94 Cupriavidus necator (strain ATCC 17699/H16/ adh, H16_A0757 Q0KDL6 DSM 428/Stanier 337) (Ralstonia eutropha) Mycobacterium tuberculosis (strain CDC 1551/ adh, MT1581 P9WQC2 Oshkosh) Staphylococcus aureus (strain MW2) adh, MW0568 Q8NXU1 Mycobacterium tuberculosis (strain ATCC 25618/ adh, Rv1530 P9WQC3 H37Rv) Staphylococcus aureus (strain N315) adh, SA0562 Q7A742 Staphylococcus aureus (strain bovine RF122/ adh, SAB0557 Q2YSX0 ET3-1) Sulfolobus acidocaldarius (strain ATCC 33909/ adh, Saci_2057 Q4J781 DSM 639/JCM 8929/NBRC 15157/NCIMB 11770) Staphylococcus aureus (strain COL) adh, SACOL0660 Q5HI63 Staphylococcus aureus (strain NCTC 8325) adh, Q2G0G1 SAOUHSC_00608 Staphylococcus aureus (strain MRSA252) adh, SAR0613 Q6GJ63 Staphylococcus aureus (strain MSSA476) adh, SAS0573 Q6GBM4 Staphylococcus aureus (strain USA300) adh, Q2FJ31 SAUSA300_0594 Staphylococcus aureus (strain Mu50/ATCC adh, SAV0605 Q99W07 700699) Staphylococcus epidermidis (strain ATCC 12228) adh, SE_0375 Q8CQ56 Staphylococcus epidermidis (strain ATCC 35984/ adh, SERP0257 Q5HRD6 RP62A) Sulfolobus solfataricus (strain ATCC 35092/DSM adh, SSO2536 P39462 1617/JCM 11322/P2) Sulfolobus tokodaii (strain DSM 16993/JCM adh, STK_25770 Q96XE0 10545/NBRC 100140/7) Anas platyrhynchos (Domestic duck) (Anas ADH1 P30350 boschas) Apteryx australis (Brown kiwi) ADH1 P49645 Ceratitis capitata (Mediterranean fruit fly) ADH1 P48814 (Tephritis capitata) Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra) ADH1 Q70UN9 Gallus gallus (Chicken) ADH1 P23991 Columba livia (Domestic pigeon) ADH1 P86883 Coturnix coturnix japonica (Japanese quail) ADH1 P19631 (Coturnix japonica) Drosophila hydei (Fruit fly) Adh1 P23236 Drosophila montana (Fruit fly) Adh1 P48586 Drosophila mettleri (Fruit fly) Adh1 P22246 Drosophila mulleri (Fruit fly) Adh1 P07161 Drosophila navojoa (Fruit fly) Adh1 P12854 Geomys attwateri (Attwater's pocket gopher) ADH1 Q9Z2M2 (Geomys bursarius attwateri) Geomys bursarius (Plains pocket gopher) ADH1 Q64413 Geomys knoxjonesi (Knox Jones's pocket gopher) ADH1 Q64415 Hordeum vulgare (Barley) ADH1 P05336 Kluyveromyces marxianus (Yeast) (Candida kefyr) ADH1 Q07288 Zea mays (Maize) ADH1 P00333 Mesocricetus auratus (Golden hamster) ADH1 P86885 Pennisetum americanum (Pearl millet) (Pennisetum ADH1 P14219 glaucum) Petunia hybrida (Petunia) ADH1 P25141 Oryctolagus cuniculus (Rabbit) ADH1 Q03505 Solanum tuberosum (Potato) ADH1 P14673 Struthio camelus (Ostrich) ADH1 P80338 Trifolium repens (Creeping white clover) ADH1 P13603 Zea luxurians (Guatemalan teosinte) (Euchlaena ADH1 Q07264 luxurians) Saccharomyces cerevisiae (strain ATCC 204508/ ADH1, ADC1, P00330 S288c) (Baker's yeast) YOL086C, O0947 Arabidopsis thaliana (Mouse-ear cress) ADH1, ADH, P06525 At1g77120, F22K20.19 Schizosaccharomyces pombe (strain 972/ATCC adh1, adh, P00332 24843) (Fission yeast) SPCC13B11.01 Drosophila lacicola (Fruit fly) Adh1, Adh-1 Q27404 Mus musculus (Mouse) Adh1, Adh-1 P00329 Peromyscus maniculatus (North American deer ADH1, ADH-1 P41680 mouse) Rattus norvegicus (Rat) Adh1, Adh-1 P06757 Drosophila virilis (Fruit fly) Adh1, Adh-1, B4M8Y0 GJ18208 Scheffersomyces stipitis (strain ATCC 58785/ ADH1, ADH2, O00097 CBS 6054/NBRC 10063/NRRL Y-11545) PICST_68558 (Yeast) (Pichia stipitis) Aspergillus flavus (strain ATCC 200026/FGSC adh1, P41747 A1120/NRRL 3357/JCM 12722/SRRC 167) AFLA_048690 Neurospora crassa (strain ATCC 24698/74-OR23- adh-1, Q9P6C8 1A/CBS 708.71/DSM 1257/FGSC 987) B17C10.210, NCU01754 Candida albicans (Yeast) ADH1, CAD P43067 Oryza sativa subsp. japonica (Rice) ADH1, DUPR11.3, Q2R8Z5 Os11g0210300, LOC_Os11g10480, OsJ_032001 Drosophila mojavensis (Fruit fly) Adh1, GI17644 P09370 Kluyveromyces lactis (strain ATCC 8585/CBS ADH1, P20369 2359/DSM 70799/NBRC 1267/NRRL Y-1140/ KLLA0F21010g WM37) (Yeast) (Candida sphaerica) Oryza sativa subsp. indica (Rice) ADH1, Q75ZX4 OsI_034290 Pongo abelii (Sumatran orangutan) (Pongo ADH1A Q5RBP7 pygmaeus abelii) Homo sapiens (Human) ADH1A, ADH1 P07327 Macaca mulatta (Rhesus macaque) ADH1A, ADH1 P28469 Pan troglodytes (Chimpanzee) ADH1B Q5R1W2 Papio hamadryas (Hamadryas baboon) ADH1B P14139 Homo sapiens (Human) ADH1B, ADH2 P00325 Homo sapiens (Human) ADH1C, ADH3 P00326 Papio hamadryas (Hamadryas baboon) ADH1C, ADH3 O97959 Ceratitis capitata (Mediterranean fruit fly) ADH2 P48815 (Tephritis capitata) Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra) ADH2 Q70UP5 Ceratitis rosa (Natal fruit fly) (Pterandrus rosa) ADH2 Q70UP6 Drosophila arizonae (Fruit fly) Adh2 P27581 Drosophila buzzatii (Fruit fly) Adh2 P25720 Drosophila hydei (Fruit fly) Adh2 P23237 Drosophila montana (Fruit fly) Adh2 P48587 Drosophila mulleri (Fruit fly) Adh2 P07160 Drosophila wheeleri (Fruit fly) Adh2 P24267 Entamoeba histolytica ADH2 Q24803 Hordeum vulgare (Barley) ADH2 P10847 Kluyveromyces marxianus (Yeast) (Candida kefyr) ADH2 Q9P4C2 Zea mays (Maize) ADH2 P04707 Oryza sativa subsp. indica (Rice) ADH2 Q4R1E8 Solanum lycopersicum (Tomato) (Lycopersicon ADH2 P28032 esculentum) Solanum tuberosum (Potato) ADH2 P14674 Scheffersomyces stipitis (strain ATCC 58785/ ADH2, ADH1, O13309 CBS 6054/NBRC 10063/NRRL Y-11545) PICST_27980 (Yeast) (Pichia stipitis) Arabidopsis thaliana (Mouse-ear cress) ADH2, ADHIII, Q96533 FDH1, At5g43940, MRH10.4 Saccharomyces cerevisiae (strain ATCC 204508/ ADH2, ADR2, P00331 S288c) (Baker's yeast) YMR303C, YM9952.05C Candida albicans (strain SC5314/ATCC MYA- ADH2, O94038 2876) (Yeast) Ca41C10.04, CaO19.12579, CaO19.5113 Oryza sativa subsp. japonica (Rice) ADH2, DUPR11.1, Q0ITW7 Os11g0210500, LOC_Os11g10510 Drosophila mojavensis (Fruit fly) Adh2, GI17643 P09369 Kluyveromyces lactis (strain ATCC 8585/CBS ADH2, P49383 2359/DSM 70799/NBRC 1267/NRRL Y-1140/ KLLA0F18260g WM37) (Yeast) (Candida sphaerica) Oryctolagus cuniculus (Rabbit) ADH2-1 O46649 Oryctolagus cuniculus (Rabbit) ADH2-2 O46650 Hordeum vulgare (Barley) ADH3 P10848 Solanum tuberosum (Potato) ADH3 P14675 Kluyveromyces lactis (strain ATCC 8585/CBS ADH3, P49384 2359/DSM 70799/NBRC 1267/NRRL Y-1140/ KLLA0B09064g WM37) (Yeast) (Candida sphaerica) Saccharomyces cerevisiae (strain ATCC 204508/ ADH3, P07246 S288c) (Baker's yeast) YMR083W, YM9582.08 Homo sapiens (Human) ADH4 P08319 Mus musculus (Mouse) Adh4 Q9QYY9 Rattus norvegicus (Rat) Adh4 Q64563 Struthio camelus (Ostrich) ADH4 P80468 Kluyveromyces lactis (strain ATCC 8585/CBS ADH4, P49385 2359/DSM 70799/NBRC 1267/NRRL Y-1140/ KLLA0F13530g WM37) (Yeast) (Candida sphaerica) Schizosaccharomyces pombe (strain 972/ATCC adh4, Q09669 24843) (Fission yeast) SPAC5H10.06c Saccharomyces cerevisiae (strain YJM789) ADH4, ZRG5, A6ZTT5 (Baker's yeast) SCY_1818 Saccharomyces cerevisiae (strain ATCC 204508/ ADH4, ZRG5, P10127 S288c) (Baker's yeast) YGL256W, NRC465 Saccharomyces pastorianus (Lager yeast) ADH5 Q6XQ67 (Saccharomyces cerevisiae x Saccharomyces eubayanus) Bos taurus (Bovine) ADH5 Q3ZC42 Equus caballus (Horse) ADH5 P19854 Mus musculus (Mouse) Adh5, Adh-2, P28474 Adh2 Rattus norvegicus (Rat) Adh5, Adh-2, P12711 Adh2 Oryctolagus cuniculus (Rabbit) ADH5, ADH3 O19053 Homo sapiens (Human) ADH5, ADHX, P11766 FDH Dictyostelium discoideum (Slime mold) adh5, Q54TC2 DDB_G0281865 Saccharomyces cerevisiae (strain ATCC 204508/ ADH5, P38113 S288c) (Baker's yeast) YBR145W, YBR1122 Homo sapiens (Human) ADH6 P28332 Peromyscus maniculatus (North American deer ADH6 P41681 mouse) Pongo abelii (Sumatran orangutan) (Pongo ADH6 Q5R7Z8 pygmaeus abelii) Rattus norvegicus (Rat) Adh6 Q5XI95 Homo sapiens (Human) ADH7 P40394 Rattus norvegicus (Rat) Adh7 P41682 Mus musculus (Mouse) Adh7, Adh-3, Q64437 Adh3 Mycobacterium tuberculosis (strain CDC 1551/ adhA, MT1911 P9WQC0 Oshkosh) Rhizobium meliloti (strain 1021) (Ensifer meliloti) adhA, RA0704, O31186 (Sinorhizobium meliloti) SMa1296 Mycobacterium tuberculosis (strain ATCC 25618/ adhA, Rv1862 P9WQC1 H37Rv) Zymomonas mobilis subsp. mobilis (strain ATCC adhA, ZMO1236 P20368 31821/ZM4/CP4) Mycobacterium bovis (strain ATCC BAA-935/ adhB, Mb0784c Q7U1B9 AF2122/97) Mycobacterium tuberculosis (strain CDC 1551/ adhB, MT0786 P9WQC6 Oshkosh) Mycobacterium tuberculosis (strain ATCC 25618/ adhB, Rv0761c, P9WQC7 H37Rv) MTCY369.06c Zymomonas mobilis subsp. mobilis (strain ATCC adhB, ZMO1596 P0DJA2 31821/ZM4/CP4) Zymomonas mobilis subsp. mobilis (strain ATCC adhB, Zmob_1541 F8DVL8 10988/DSM 424/LMG 404/NCIMB 8938/ NRRL B-806/ZM1) Mycobacterium tuberculosis (strain CDC 1551/ adhD, MT3171 P9WQB8 Oshkosh) Mycobacterium tuberculosis (strain ATCC 25618/ adhD, Rv3086 P9WQB9 H37Rv) Clostridium acetobutylicum (strain ATCC 824/ adhE, aad, P33744 DSM 792/JCM 1419/LMG 5710/VKM B- CA_P0162 1787) Escherichia coli (strain K12) adhE, ana, b1241, P0A9Q7 JW1228 Escherichia coli O157:H7 adhE, Z2016, P0A9Q8 ECs1741 Rhodobacter sphaeroides (strain ATCC 17023/ adhI, P72324 2.4.1/NCIB 8253/DSM 158) RHOS4_11650, RSP_2576 Oryza sativa subsp. indica (Rice) ADHIII, A2XAZ3 OsI_009236 Escherichia coli (strain K12) adhP, yddN, P39451 b1478, JW1474 Geobacillus stearothermophilus (Bacillus adhT P12311 stearothermophilus) Emericella nidulans (strain FGSC A4/ATCC alcA, AN8979 P08843 38163/CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Emericella nidulans (strain FGSC A4/ATCC alc, AN3741 P54202 38163/CBS 112.46/NRRL 194/M139) (Aspergillus nidulans) Emericella nidulans (strain FGSC A4/ATCC alcC, adh3, P07754 38163/CBS 112.46/NRRL 194/M139) AN2286 (Aspergillus nidulans) Arabidopsis thaliana (Mouse-ear cress) At1g22430, Q9SK86 F12K8.22 Arabidopsis thaliana (Mouse-ear cress) At1g22440, Q9SK87 F12K8.21 Arabidopsis thaliana (Mouse-ear cress) At1g32780, A1L4Y2 F6N18.16 Arabidopsis thaliana (Mouse-ear cress) At1g64710, Q8VZ49 F13O11.3 Arabidopsis thaliana (Mouse-ear cress) At4g22110, Q0V7W6 F1N20.210 Arabidopsis thaliana (Mouse-ear cress) At5g24760, Q8LEB2 T4C12_30 Arabidopsis thaliana (Mouse-ear cress) At5g42250, Q9FH04 K5J14.5 Zea mays (Maize) FDH P93629 Drosophila melanogaster (Fruit fly) Fdh, gfd, ODH, P46415 CG6598 Bacillus subtilis (strain 168) gbsB, BSU31050 P71017 Caenorhabditis elegans H24K24.3 Q17335 Oryza sativa subsp. japonica (Rice) Os02g0815500, Q0DWH1 LOC_Os02g57040, OsJ_008550, P0643F09.4 Mycobacterium tuberculosis (strain ATCC 25618/ Rv1895 O07737 H37Rv) Caenorhabditis elegans sodh-1, K12G11.3 Q17334 Caenorhabditis elegans sodh-2, K12G11.4 O45687 Pseudomonas sp. terPD P33010 Escherichia coli (strain K12) yiaY, b3589, P37686 JW5648 Moraxella sp. (strain TAE123) P81786 Alligator mississippiensis (American alligator) P80222 Catharanthus roseus (Madagascar periwinkle) P85440 (Vinca rosea) Gadus morhua subsp. callarias (Baltic cod) (Gadus P26325 callarias) Naja naja (Indian cobra) P80512 Pisum sativum (Garden pea) P12886 Pelophylax perezi (Perez's frog) (Rana perezi) P22797 Saara hardwickii (Indian spiny-tailed lizard) P25405 (Uromastyx hardwickii) Saara hardwickii (Indian spiny-tailed lizard) P25406 (Uromastyx hardwickii) Equus caballus (Horse) P00327 Equus caballus (Horse) P00328 Geobacillus stearothermophilus (Bacillus P42328 stearothermophilus) Gadus morhua (Atlantic cod) P81600 Gadus morhua (Atlantic cod) P81601 Myxine glutinosa (Atlantic hagfish) P80360 Octopus vulgaris (Common octopus) P81431 Pisum sativum (Garden pea) P80572 Saara hardwickii (Indian spiny-tailed lizard) P80467 (Uromastyx hardwickii) Scyliorhinus canicula (Small-spotted catshark) P86884 (Squalus canicula) Sparus aurata (Gilthead sea bream) P79896

In some embodiments, an α-dioxygenase is used to catalyze the conversion of a fatty acid to a fatty aldehyde (Hamberg et al. 2005). Alpha-dioxygenases catalyze the conversion of a C_(n) fatty acid to a C_(n-1) aldehyde and may serve as an alternative to both ADH and AOX for fatty aldehyde production if a fatty acid is used as a biotransformation substrate. Due to the chain shortening of the dioxygenase reaction, this route requires a different synthesis pathway compared to the ADH and AOX routes. Biotransformations of E. coli cells expressing a rice α-dioxygenase exhibited conversion of C10, C12, C14 and C16 fatty acids to the corresponding C_(n-1) aldehydes. With the addition of the detergent Triton X 100, 3.7 mM of pentadecanal (0.8 g/L) was obtained after 3 hours from hexadecanoic acid with 74% conversion (Kaehne et al. 2011). Exemplary α-dioxygenases are shown in Table 10.

TABLE 10 Exemplary alpha-dioxygenases Entry Organism Gene names Q9SGH6 Arabidopsis thaliana (Mouse-ear cress) DOX1 DIOX1 PADOX-1 PIOX At3g01420 T13O15.6 Q9C9U3 Arabidopsis thaliana (Mouse-ear cress) DOX2 DIOX2 At1g73680 F25P22.10 P14550 Homo sapiens (Human) AKR1A1 ALDR1 ALR Q69EZ9 Solanum lycopersicum (Tomato) LOC543896 (Lycopersicon esculentum) Q5WM33 Solanum lycopersicum (Tomato) alpha-DOX2 (Lycopersicon esculentum) Q69F00 Solanum lycopersicum (Tomato) (Lycopersicon esculentum) D7LAG3 Arabidopsis lyrata subsp. lyrata (Lyre- ALPHA-DOX1 leaved rock-cress) ARALYDRAFT_317048 D8LJL3 Ectocarpus siliculosus (Brown alga) DOX Esi_0026_0091 E3U9P5 Nicotiana attenuata (Coyote tobacco) adox2

An enzyme's total turnover number (or TTN) refers to the maximum number of molecules of a substrate that the enzyme can convert before becoming inactivated. In general, the TTN for the hydroxylases and other enzymes used in the methods of the disclosure range from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more.

When whole cells expressing a hydroxylase are used to carry out a hydroxylation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo hydroxylation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol·g_(cdw) ⁻¹, wherein gnaw is the mass of cell dry weight in grams. When whole cells expressing a hydroxylase are used to carry out a hydroxylation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g g_(cdw) ⁻¹ h⁻¹). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g·g_(cdw) ⁻¹ h⁻¹, wherein gnaw is the mass of cell dry weight in grams.

The TTN for heme enzymes, in particular, typically ranges from about 1 to about 100,000 or higher. For example, the TTN can be from about 1 to about 1,000, or from about 1,000 to about 10,000, or from about 10,000 to about 100,000, or from about 50,000 to about 100,000, or at least about 100,000. In particular embodiments, the TTN can be from about 100 to about 10,000, or from about 10,000 to about 50,000, or from about 5,000 to about 10,000, or from about 1,000 to about 5,000, or from about 100 to about 1,000, or from about 250 to about 1,000, or from about 100 to about 500, or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, 100,000, or more. In certain embodiments, the variant or chimeric heme enzymes of the present disclosure have higher TTNs compared to the wild-type sequences. In some instances, the variant or chimeric heme enzymes have TTNs greater than about 100 (e.g., at least about 100, 150, 200, 250, 300, 325, 350, 400, 450, 500, or more) in carrying out in vitro hydroxylation reactions. In other instances, the variant or chimeric heme enzymes have TTNs greater than about 1000 (e.g., at least about 1000, 2500, 5000, 10,000, 25,000, 50,000, 75,000, 100,000, or more) in carrying out in vivo whole cell hydroxylation reactions.

When whole cells expressing a heme enzyme are used to carry out a hydroxylation reaction, the turnover can be expressed as the amount of substrate that is converted to product by a given amount of cellular material. In general, in vivo hydroxylation reactions exhibit turnovers from at least about 0.01 to at least about 10 mmol·g_(cdw) ⁻¹, wherein gnaw is the mass of cell dry weight in grams. For example, the turnover can be from about 0.1 to about 10 mmol·g_(cdw) ⁻¹, or from about 1 to about 10 mmol·g_(cdw) ⁻¹, or from about 5 to about 10 mmol·g_(cdw) ⁻¹, or from about 0.01 to about 1 mmol·g_(cdw) ⁻¹, or from about 0.01 to about 0.1 mmol·g_(cdw) ⁻¹, or from about 0.1 to about 1 mmol·g_(cdw) ⁻¹, or greater than 1 mmol·g_(cdw) ⁻¹. The turnover can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10 mmol·g_(cdw) ⁻¹.

When whole cells expressing a heme enzyme are used to carry out a hydroxylation reaction, the activity can further be expressed as a specific productivity, e.g., concentration of product formed by a given concentration of cellular material per unit time, e.g., in g/L of product per g/L of cellular material per hour (g·g_(cdw) ⁻¹ h⁻¹). In general, in vivo hydroxylation reactions exhibit specific productivities from at least about 0.01 to at least about 0.5 g·g_(cdw) ⁻¹ h⁻¹, wherein gnaw is the mass of cell dry weight in grams. For example, the specific productivity can be from about 0.01 to about 0.1 g·g_(cdw) ⁻¹ h⁻¹, or from about 0.1 to about 0.5 g·g_(cdw) ⁻¹ h⁻¹, or greater than 0.5 g·g_(cdw) ⁻¹ h⁻¹. The specific productivity can be about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or about 0.5 g·g_(cdw) ⁻¹ h⁻¹.

In certain embodiments, mutations can be introduced into the target gene using standard cloning techniques (e.g., site-directed mutagenesis) or by gene synthesis to produce the hydroxylases (e.g., cytochrome P450 variants) of the present disclosure. The mutated gene can be expressed in a host cell (e.g., bacterial cell) using an expression vector under the control of an inducible promoter or by means of chromosomal integration under the control of a constitutive promoter. Hydroxylation activity can be screened in vivo or in vitro by following product formation by GC or HPLC as described herein.

The expression vector comprising a nucleic acid sequence that encodes a heme enzyme of the disclosure can be a viral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage (e.g., a bacteriophage P1-derived vector (PAC)), a baculovirus vector, a yeast plasmid, or an artificial chromosome (e.g., bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a mammalian artificial chromosome (MAC), and human artificial chromosome (HAC)). Expression vectors can include chromosomal, non-chromosomal, and synthetic DNA sequences. Equivalent expression vectors to those described herein are known in the art and will be apparent to the ordinarily skilled artisan.

The expression vector can include a nucleic acid sequence encoding a heme enzyme that is operably linked to a promoter, wherein the promoter comprises a viral, bacterial, archaeal, fungal, insect, or mammalian promoter. In certain embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In other embodiments, the promoter is a tissue-specific promoter or an environmentally regulated or a developmentally regulated promoter.

It is to be understood that affinity tags may be added to the N- and/or C-terminus of a heme enzyme expressed using an expression vector to facilitate protein purification. Non-limiting examples of affinity tags include metal binding tags such as His6-tags and other tags such as glutathione S-transferase (GST).

Non-limiting expression vectors for use in bacterial host cells include pCWori, pET vectors such as pET22 (EMD Millipore), pBR322 (ATCC37017), pQE™ vectors (Qiagen), pBluescript™ vectors (Stratagene), pNH vectors, lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia), pRSET, pCR-TOPO vectors, pET vectors, pSyn_1 vectors, pChlamy_1 vectors (Life Technologies, Carlsbad, Calif.), pGEM1 (Promega, Madison, Wis.), and pMAL (New England Biolabs, Ipswich, Mass.). Non-limiting examples of expression vectors for use in eukaryotic host cells include pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia), pcDNA3.3, pcDNA4/TO, pcDNA6/TR, pLenti6/TR, pMT vectors (Life Technologies), pKLAC1 vectors, pKLAC2 vectors (New England Biolabs), pQE™ vectors (Qiagen), BacPak baculoviral vectors, pAdeno-X™ adenoviral vectors (Clontech), and pBABE retroviral vectors. Any other vector may be used as long as it is replicable and viable in the host cell.

The host cell can be a bacterial cell, an archaeal cell, a fungal cell, a yeast cell, an insect cell, or a mammalian cell.

Suitable bacterial host cells include, but are not limited to, BL21 E. coli, DE3 strain E. coli, E. coli M15, DH5α, DH10β, HB101, T7 Express Competent E. coli (NEB), B. subtilis cells, Pseudomonas fluorescens cells, and cyanobacterial cells such as Chlamydomonas reinhardtii cells and Synechococcus elongates cells. Non-limiting examples of archaeal host cells include Pyrococcus furiosus, Metallosphera sedula, Thermococcus litoralis, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Pyrococcus abyssi, Sulfolobus solfataricus, Pyrococcus woesei, Sulfolobus shibatae, and variants thereof. Fungal host cells include, but are not limited to, yeast cells from the genera Saccharomyces (e.g., S. cerevisiae), Pichia (P. Pastoris), Candida (C. tropicalis), Kluyveromyces (e.g., K. lactis), Hansenula and Yarrowia, and filamentous fungal cells from the genera Aspergillus, Trichoderma, and Myceliophthora. Suitable insect host cells include, but are not limited to, Sf9 cells from Spodoptera frugiperda, Sf21 cells from Spodoptera frugiperda, Hi-Five cells, BTI-TN-5B1-4 Trichophusia ni cells, and Schneider 2 (S2) cells and Schneider 3 (S3) cells from Drosophila melanogaster. Non-limiting examples of mammalian host cells include HEK293 cells, HeLa cells, CHO cells, COS cells, Jurkat cells, NS0 hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell.

In certain embodiments, the present disclosure provides heme enzymes such as the P450 variants described herein that are active hydroxylation catalysts inside living cells. As a non-limiting example, bacterial cells (e.g., E. coli) can be used as whole cell catalysts for the in vivo hydroxylation reactions of the present disclosure. In some embodiments, whole cell catalysts containing P450 enzymes with the equivalent C400X mutation are found to significantly enhance the total turnover number (TTN) compared to in vitro reactions using isolated P450 enzymes.

Biohydroxylation Reaction Conditions

The methods of the disclosure include forming reaction mixtures that contain the hydroxylases described herein. The hydroxylases can be, for example, purified prior to addition to a reaction mixture or secreted by a cell present in the reaction mixture. The reaction mixture can contain a cell lysate including the enzyme, as well as other proteins and other cellular materials. Alternatively, a hydroxylase can catalyze the reaction within a cell expressing the hydroxylase. Any suitable amount of hydroxylase can be used in the methods of the disclosure. In general, hydroxylation reaction mixtures contain from about 0.01 weight % (wt %) to about 100 wt % hydroxylase with respect to the hydrocarbon substrate. The reaction mixtures can contain, for example, from about 0.01 wt % to about 0.1 wt % hydroxylase, or from about 0.1 wt % to about 1 wt % hydroxylase, or from about 1 wt % to about 10 wt % hydroxylase, or from about 10 wt % to about 100 wt % hydroxylase. The reaction mixtures can contain from about 0.05 wt % to about 5 wt % hydroxylase, or from about 0.05 wt % to about 0.5 wt % hydroxylase. The reaction mixtures can contain about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, or about 3 wt % hydroxylase. One of skill in the art will understand how to convert wt % values to mol % values with respect to the hydroxylase and/or substrate concentrations set forth herein.

If the hydroxylase catalyses the reaction within a cell expressing the hydroxylase then any suitable amount of cells can be used in the methods of the disclosure. In general, hydroxylation whole-cell reaction mixtures contain from about 1 weight % to about 10,000 wt % of cells on a cell dry weight basis with respect to the hydrocarbon substrate. The whole-cell reaction mixtures can contain, for example, from about 1 wt % to about 10 wt % cells, or from about 10 wt % to about 100 wt % cells, or from about 100 wt % to about 1000 wt % cells, or from about 1000 wt % cells to about 2500 wt % cells, or from about 2500 wt % cells to about 5000 wt % cells, or from about 5000 wt % cells to about 7500 wt % cells, or from about 7500 wt % cells to about 10000 wt % cells with respect to the hydrocarbon substrate. The whole-cell reaction mixtures can contain from about 2 wt % to about 1000 wt % cells, or from about 5 wt % to about 500 wt % cells with respect to the hydrocarbon substrate. The whole-cell reaction mixtures can contain about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 wt % cells with respect to the hydrocarbon substrate.

The concentration of a saturated or unsaturated hydrocarbon substrate is typically in the range of from about 100 μM to about 1 M. The concentration can be, for example, from about 100 μM to about 1 mM, or about from 1 mM to about 100 mM, or from about 100 mM to about 500 mM, or from about 500 mM to 1 M. The concentration can be from about 500 μM to about 500 mM, 500 μM to about 50 mM, or from about 1 mM to about 50 mM, or from about 15 mM to about 45 mM, or from about 15 mM to about 30 mM. The concentration of the saturated or unsaturated hydrocarbon substrate can be, for example, about 100, 200, 300, 400, 500, 600, 700, 800, or 900 μM. The concentration of the saturated or unsaturated hydrocarbon substrate can be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM.

Reaction mixtures can contain additional reagents. As non-limiting examples, the reaction mixtures can contain buffers (e.g., 2-(N-morpholino)ethanesulfonic acid (MES), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 3-morpholinopropane-1-sulfonic acid (MOPS), 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate, sodium phosphate, phosphate-buffered saline, sodium citrate, sodium acetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide, dimethylformamide, ethanol, methanol, isopropanol, glycerol, tetrahydrofuran, acetone, acetonitrile, and acetic acid), salts (e.g., NaCl, KCl, CaCl₂, and salts of Mn²⁺ and Mg²⁺), denaturants (e.g., urea and guandinium hydrochloride), detergents (e.g., sodium dodecylsulfate and Triton-X 100), chelators (e.g., ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 2-({2-[Bis(carboxymethyl)amino]ethyl} (carboxymethyl)amino)acetic acid (EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA)), sugars (e.g., glucose, sucrose, and the like), and reducing agents (e.g., sodium dithionite, NADPH, dithiothreitol (DTT), β-mercaptoethanol (BME), and tris(2-carboxyethyl)phosphine (TCEP)). Buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents can be used at any suitable concentration, which can be readily determined by one of skill in the art. In general, buffers, cosolvents, salts, denaturants, detergents, chelators, sugars, and reducing agents, if present, are included in reaction mixtures at concentrations ranging from about 1 μM to about 1 M. For example, a buffer, a cosolvent, a salt, a denaturant, a detergent, a chelator, a sugar, or a reducing agent can be included in a reaction mixture at a concentration of about 1 μM, or about 10 μM, or about 100 μM, or about 1 mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 100 mM, or about 250 mM, or about 500 mM, or about 1 M. Cosolvents, in particular, can be included in the reaction mixtures in amounts ranging from about 1% v/v to about 75% v/v, or higher. A co-solvent can be included in the reaction mixture, for example, in an amount of about 5, 10, 20, 30, 40, or 50% (v/v).

Reactions are conducted under conditions sufficient to catalyze the formation of a hydroxylation product. The reactions can be conducted at any suitable temperature. In general, the reactions are conducted at a temperature of from about 4° C. to about 40° C. The reactions can be conducted, for example, at about 25° C. or about 37° C. The reactions can be conducted at any suitable pH. In general, the reactions are conducted at a pH of from about 3 to about 10. The reactions can be conducted, for example, at a pH of from about 6.5 to about 9. The reactions can be conducted for any suitable length of time. In general, the reaction mixtures are incubated under suitable conditions for anywhere between about 1 minute and several hours. The reactions can be conducted, for example, for about 1 minute, or about 5 minutes, or about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12 hours, or about 24 hours, or about 48 hours, or about 72 hours, or about 96 hours, or about 120 hours, or about 144 hours, or about 168 hours, or about 192 hours. In general, reactions are conducted under aerobic conditions. In some embodiments, the solvent forms a second phase, and the hydroxylation occurs in the aqueous phase. In some embodiments, the hydroxylases is located in the aqueous layer whereas the substrates and/or products occur in an organic layer. Other reaction conditions may be employed in the methods of the disclosure, depending on the identity of a particular hydroxylase, or olefinic substrate.

Reactions can be conducted in vivo with intact cells expressing a hydroxylase of the disclosure. The in vivo reactions can be conducted with any of the host cells used for expression of the hydroxylases, as described herein. A suspension of cells can be formed in a suitable medium supplemented with nutrients (such as mineral micronutrients, glucose and other fuel sources, and the like). Hydroxylation yields from reactions in vivo can be controlled, in part, by controlling the cell density in the reaction mixtures. Cellular suspensions exhibiting optical densities ranging from about 0.1 to about 50 at 600 nm can be used for hydroxylation reactions. Other densities can be useful, depending on the cell type, specific hydroxylases, or other factors.

Pheromones, Precursors, Positional Isomers, and Analogs Comprising More than One C═C Double Bond

In some embodiments, an olefinic product described herein can have more than one carbon-carbon (C═C) double bond. In some embodiments, such olefinic products can be used to synthetically derive pheromones with more than one double bond.

In some embodiments, conjugated and unconjugated alkenes can be biohydroxylated to generate corresponding conjugated and unconjugated alkenols as illustrated in Scheme 11 below.

In some embodiments, biohydroxylation occurs on the terminal carbon an m-end of a carbon-carbon double bond in an unsaturated hydrocarbon substrate to produce first synthetically derived insect pheromone having a chemical structure corresponding to the chemical structure of a naturally occurring insect pheromone produced by the target insect. In some embodiments, biohydroxylation occurs on a terminal carbon of an n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate a positional isomer of said first synthetically derived insect pheromone. In some embodiments, biohydroxylation occurs on a subterminal carbon on the m-end or the n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate thereby forming an i-end, wherein the i-end comprises a terminal carbon of the unsaturated hydrocarbon substrate. In some embodiments, biohydroxylation and subsequent oxidation produces a pheromone or precursor that is over-oxidized, e.g., by hydroxylation of both terminal carbons (i.e., the m-end and the n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate) and/or oxidation to a carboxylic acid.

The substrate, the pheromone, the positional isomer, and the analog can have any suitable combination of subscripts a, b, c, d, e, i, m, and n. In some embodiments, a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, or e is 1. In some embodiments, m and n are integers independently selected from 1 to 15. In some embodiments, m, n, and i are integers independently selected from 1 to 15. In some embodiments, b and d are integers independently selected from 1 to 10. In some embodiments, the sum of a, b, c, d, e, m, and n is an integer that results in a total number of carbons from 6 to 20. In some embodiments, the sum of a, b, c, d, e, i, m, and n is an integer that results in a total number of carbons from 6 to 20. In some embodiments, each R is independently —OH, ═O, or —OAc. In some embodiments, each R′ is independently —OH, ═O, —OAc, or —OOH.

Pheromone Compositions

In some embodiments, the present disclosure provides for a pheromone and its positional isomer. For example, in some embodiments, the present disclosure provides for the synthesis of (E/Z)-hexadecen-1-al. Depending on the synthetic route and starting material, embodiments described herein, a variety of isomers of Z-hexadecen-1-al can be synthesized. Accordingly, a pheromone composition prepared as described herein can include a mixture of two or more of the following isomers: Z-hexadec-2-en-1-al, Z-hexadec-3-en-1-al, Z-hexadec-4-en-1-al, Z-hexadec-5-en-1-al, Z-hexadec-6-en-1-al, Z-hexadec-7-en-1-al, Z-hexadec-8-en-1-al, Z-hexadec-9-en-1-al, Z-hexadec-10-en-1-al, Z-hexadec-11-en-1-al, Z-hexadec-12-en-1-al, Z-hexadec-13-en-1-al, Z-hexadec-14-en-1-al, and Z-hexadec-15-en-1-al. Thus, in some embodiments, a pheromone composition as described herein can include at least one isomer of a natural pheromone or a mixture of isomers.

In some embodiments, the isomer is a positional isomer. The positional isomer produced using the methodology disclosed herein occurs via biohydroxylation of a location on the carbon skeleton which is different from location required to produce the natural pheromone for an insect. Accordingly, in some embodiments, a pheromone composition as described herein can include a natural pheromone produced by an insect and least one positional isomer of the natural pheromone. In some embodiments, the positional isomer is not produced by the insect whose behavior is modified by the pheromone composition. Thus, in some embodiments, the pheromone composition can include a first insect pheromone having a chemical structure of an insect sex pheromone produced by a member of the order Lepidoptera and a positional isomer of said first insect pheromone. In one such embodiment, the positional isomer is not produced by a member of the order Lepidoptera.

Mixtures of a pheromone with its positional isomer, as disclosed herein, can be used modulate the behavior of Lepidopteran species in a controllable or tunable manner. Although positional isomers of a pheromone, which are contained in various compositions of the disclosure, may not be emitted by a female Lepidoptera, its presence in mixtures with the authentic pheromone elicits a mating response from male Lepidoptera. The mating response of the male insects differ with different ratios of the positional isomer to the authentic pheromone, which indicates that the use of pheromones in mixtures of its positional isomers enables modulation of a male insect response that cannot be obtained with pure pheromone alone. For example, mixtures of (Z)-5-hexadecenal with the natural (Z)-11hexadecenal are able to elicit a mating response from H. zea males, even though (Z)-11-hexadecenal is emitted by the female Lepidoptera. The addition of (Z)-9-hexadecenal to mixtures of (Z)-5-hexadecenal and (Z)-11-hexadecenal may also act an as insect pheromone attractant. Accordingly, embodiments of the disclosure provide for mixtures of a natural pheromone with its positional isomer to elicit altered insect responses. The elicited response can be tuned depending on the ratio of the positional isomer to the natural pheromone. Thus, the amount of the positional isomer present in the mixture can be used to attenuate the mating response of an insect, e.g., male Lepidoptera, thereby eliciting a response which would not be possible with the natural pheromone.

In an exemplary embodiment, the pheromone compositions can include at least one synthetically derived natural pheromone and its synthetically derived positional isomer. In some embodiments, the pheromone composition includes Z-11-hexadecenal and it positional isomer Z-5-hexadecenal. In some embodiments, the pheromone composition includes a synthetically derived natural blend of Z-11-hexadecenal/Z-9-hexadecenal and the synthetically derived positional isomer of Z-11-hexadecenal—Z-5-hexadecenal, which is not produced by the target insect. In a further embodiment, the pheromone composition can also include the synthetically derived positional isomer of Z-9-hexadecenal—Z-7-hexadecenal, which is not produced by the target insect.

Thus, in some such embodiments, the pheromone composition can include compounds selected from the group consisting of Z-11-hexadecenal, Z-5-hexadecenal, Z-9-hexadecenal, Z-7-hexadecenal and combinations thereof. In other exemplary embodiments, the pheromone composition can include at least one of following combinations of synthetically derived natural pheromones and its positional isomer: Z-11-hexadecenal and Z-5-hexadecenal, or Z-9-hexadecenal and Z-7-hexadecenal.

In some embodiments, the pheromone composition includes a mixture of Z-11-hexadecenal and Z-5-hexadecenal. In some such embodiments, the percent of Z-11-hexadecenal to the percent of Z-5-hexadecenal in the composition is about 99.9% to about 0.1%, about 99.8% to about 0.2%, about 99.7% to about 0.3%, about 99.6% to about 0.4%, about 99.5% to about 0.5%, about 99.4% to about 0.6%, about 99.3% to about 0.7%, about 99.2% to about 0.8%, or about 99.1% to about 0.9%, including all values and subranges in between. In other embodiments, the ratio of Z-11-hexadecenal to Z-5-hexadecenal in the composition is about 99% to about 1.0%, 98% to about 2.0%, about 97% to about 3.0%, about 96% to about 4.0%, about 94% to about 6.0%, about 93% to about 7.0%, about 92% to about 8.0%, about 91% to about 9%, about 90% to about 10%, about 85% to about 15%, about 80% to about 20%, about 75% to about 25%, about 70% to about 30%, about 65% to about 35%, about 60% to about 40%, about 55% to about 45%, about 50% to about 50%, including all values and subranges in between.

In some such embodiments, the percent of Z-5-hexadecenal to the percent of Z-11-hexadecenal in the composition is about 99.9% to about 0.1%, about 99.8% to about 0.2%, about 99.7% to about 0.3%, about 99.6% to about 0.4%, about 99.5% to about 0.5%, about 99.4% to about 0.6%, about 99.3% to about 0.7%, about 99.2% to about 0.8%, or about 99.1% to about 0.9%, include all values and subranges in between. In some such embodiments, the percent of Z-5-hexadecenal to the percent of Z-11-hexadecenal in the composition is about 99% to about 1.0%, about 98% to about 2.0%, about 97% to about 3.0%, about 96% to about 4.0%, about 94% to about 6.0%, about 93% to about 7.0%, about 92% to about 8.0%, about 91% to about 9%, about 90% to about 10%, about 85% to about 15%, about 80% to about 20%, about 75% to about 25%, about 70% to about 30%, about 65% to about 35%, about 60% to about 40%, about 55% to about 45%, about 50% to about 50%, including all values and subranges in between.

In some embodiments, Z-11-hexadecenal is present in the composition at a percent of from about 99% mol to about 1 mol %, about 95 mol % to about 5 mol %, about 90 mol % to about 10 mol %, about 85 mol % to about 15 mol %, about 80 mol % to about 20 mol %, about 75 mol % to about 25 mol %, about 70 mol % to about 30 mol %, about 65 mol % to about 35 mol %, about 60 mol % to about 40 mol %, about 55 mol % to about 45 mol %, including all values and subranges in between. In some embodiments, Z-11-hexadecenal is present in the composition at a percent of about 97 mol % or less.

In some embodiments, the Z-11-hexadecenal is present in the composition in an amount of from about 99.9% w/w to about 0.1% w/w, about 99% to about 1% w/w, about 98% w/w to about 2% w/w, about 97% w/w to about 3% w/w, about 96% w/w to about 4% w/w, about 95% w/w to about 5% w/w, about 90% w/w to about 10% w/w, about 80% w/w to about 20% w/w, about 70% w/w to about 30% w/w, about 60% w/w to about 40% w/w, or about 50% w/w. In some embodiments, Z-11-hexadecenal is present in the composition at a percent of about 97% or less.

In some embodiments, Z-5-hexadecenal is present at a percent of from about 99.9 mol % to about 0.1 mol %, about 99 mol % to about 1 mol %, about 95 mol % to about 5 mol %, about 90 mol % to about 10 mol %, about 85 mol % to about 15 mol %, about 80 mol % to about 20 mol %, about 75 mol % to about 25 mol %, about 70 mol % to about 30 mol %, about 65 mol % to about 35 mol %, about 60 mol % to about 40 mol %, about 55 mol % to about 45 mol %, including all values and subranges in between. In other embodiments, Z-5-hexadecenal is present in the composition at percent of about 100 mol % or less, about 50 mol % or less, or about 5 mol % or less.

In some embodiments, the Z-5-hexadecenal is present in the composition in an amount of from about 99.9% w/w to about 0.1% w/w, 99% w/w to about 1% w/w, about 98% w/w to about 2% w/w, about 97% w/w to about 3% w/w, about 96% w/w to about 4% w/w, about 95% w/w to about 5% w/w, about 90% w/w to about 10% w/w, about 80% w/w to about 20% w/w, about 70% w/w to about 30% w/w, about 60% w/w to about 40% w/w, or about 50% w/w. In other embodiments, Z-5-hexadecenal is present in in the composition in an amount about 100% w/w or less, about 50% w/w or less, about 5% w/w or less, or about 0.5% or less.

In some embodiments, the Z-9-hexadecenal is present in the composition at a percent of from about 99.9 mol % to about 0.1 mol %, about 99 mol % to about 1 mol %, about 95 mol % to about 5 mol %, about 90 mol % to about 10 mol %, about 85 mol % to about 15 mol %, about 80 mol % to about 20 mol %, about 75 mol % to about 25 mol %, about 70 mol % to about 30 mol %, about 65 mol % to about 35 mol %, about 60 mol % to about 40 mol %, about 55 mol % to about 45 mol %, including all values and subranges in between. In some embodiments, Z-9-hexadecenal is present in the composition at less than or equal to about 50 mol %, less than or equal to about 40 mol %, less than or equal to about 30 mol %, less than or equal to about 25 mol %, less than or equal to about 20 mol %, less than or equal to about 15 mol %, or less than or equal to about 10 mol %. In some embodiments, Z-9-hexadecenal is present at less than or equal to about 10 mol %, less than or equal to about 9 mol %, less than or equal to about 8 mol %, less than or equal to about 7 mol %, less than or equal to about 6 mol %, less than or equal to about 5 mol %, less than or equal to about 4 mol %, less than or equal to about 3 mol %, less than or equal to about 2 mol %, or less than or equal to 1 mol %. In some embodiments, the Z-9-hexadecenal is present at about 3 mol % or less, about 2.5 mol %, at about 2 mol %, at about 1.5 mol %, at about 1 mol %, or at about 0.5 mol % or less.

In some embodiments, the Z-9-hexadecenal is present in the composition in an about amount of from about 99.9% w/w to about 0.1% w/w, about 99% w/w to about 1% w/w, about 98% to about 2% w/w, about 97% to about 3% w/w, about 96% to about 4% w/w, about 95% to about 5% w/w, about 90% to about 10% w/w, about 80% to about 20% w/w, about 70% to about 30% w/w, about 60% to about 40% w/w, or about 50% w/w. In some embodiments, the Z-9-hexadecenal is present in the composition in an amount of 3% w/w or less.

In some embodiments, the Z-7-hexadecenal is present at a percent of from about 99.9 mol % to about 0.1 mol %, about 99 mol % to about 1 mol %, about 95 mol % to about 5 mol %, about 90 mol % to about 10 mol %, about 85 mol % to about 15 mol %, about 80 mol % to about 20 mol %, about 75 mol % to about 25 mol %, about 70 mol % to about 30 mol %, about 65 mol % to about 35 mol %, about 60 mol % to about 40 mol %, about 55 mol % to about 45 mol %, including all values and subranges in between. In some embodiments, Z-9-hexadecenal is preset less than or equal to about 50 mol %, less than or equal to about 40 mol %, less than or equal to about 30 mol %, less than or equal to about 25 mol %, less than or equal to about 20 mol %, less than or equal to about 15 mol %, or less than or equal to about 10 mol %. In some embodiments, Z-7-hexadecenal is preset less than or equal to about 10 mol %, less than or equal to about 9 mol %, less than or equal to about 8 mol %, less than or equal to about 7 mol %, less than or equal to about 6 mol %, less than or equal to about 5 mol %, less than or equal to about 4 mol %, less than or equal to about 3 mol %, less than or equal to about 2 mol %, or less than or equal to 1 mol %. In some embodiments, the Z-7-hexadecenal is present at about 3 mol %, about 2.5 mol %, at about 2 mol %, at about 1.5 mol %, at about 1 mol %, or at about 0.5 mol % or less.

In some embodiments, the Z-7-hexadecenal is present in the composition in an amount of from about 99.9% w/w to about 0.1% w/w, about 99% w/w to about 1% w/w, about 98% w/w to about 2% w/w, about 97% w/w to about 3% w/w, about 96% w/w to about 4% w/w, about 95% w/w to about 5% w/w, about 90% w/w to about 10% w/w, about 80% w/w to about 20% w/w, about 70% w/w to about 30% w/w, about 60% w/w to about 40% w/w, or about 50% w/w.

In some embodiments, the pheromone composition comprises about 97% Z-11-hexadecenal and about 3% Z-9-hexadecenal. In further embodiments, Z-5-hexadecenal is added to the composition comprising 97/3 Z-11-hexadecenal to Z-9-hexadecenal such that the Z-5-hexadecenal constitutes about 0.5 mol %, about 1 mol %, about 5 mol %, about 10 mol %, about 15 mol %, about 20 mol %, about 25 mol %, about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 50 mol %, about 55 mol %, about 60 mol %, about 65 mol %, about 70 mol %, about 75 mol %, about 80 mol %, about 85 mol %, about 90 mol %, about 95 mol % or about 99 mol %, of the resulting composition.

By varying the ratio of the synthetically derived sex pheromone to its positional isomer, embodiments described herein create a tunable pheromone composition which can be used to modulate the response of the target insect species. In some embodiments, the ratio of the sex pheromone to the positional isomer can varied by selecting and/or engineering the biocatalyst. The insect that is “attracted” to the compositions taught herein may, or may not, physically contact a locus containing said pheromone composition. That is, in some aspects, the compositions taught herein are able to attract a given insect within a close proximity to a locus containing the disclosed pheromone compositions, but do not entice said insect to physically contact the locus. However, in other aspects, the compositions taught herein do entice and/or attract an insect to physically come into contact with a locus containing said pheromone compositions. In this way, inter alia, the pheromone compositions taught herein are highly “tunable” and are able to modulate the behavior (e.g., degree of attracting an insect) of an insect to a high degree, which is not associated with pheromone compositions of the prior art (i.e., compositions including only the natural pheromone). Accordingly, the pheromone compositions of the present disclosure are able to modulate the degree to which an insect is attracted along a continuous scale, depending upon, among other things, the ratio of natural pheromone to its positional isomer.

Agricultural Compositions

As described above, a variety of pheromones can be synthesized according to the MBO or MBE method. Further, utilization of the aforementioned synthesis methods can produce positional isomers of said pheromones via biohydroxylation of an alternative location of the carbon skeleton. The pheromone and its positional isomer, prepared according to these methods, can be formulated for use in compositions which modify the behavior of insects, e.g., by applying the pheromone composition to a locus thereby attracting a target insect. Pheromone compositions can contain at least one pheromone and optionally adjuvants and other compounds provided that such compounds do not substantially interfere with the activity of the composition.

In some embodiments, the agricultural compositions of the present disclosure may include, but are not limited to: wetters, compatibilizing agents (also referred to as “compatibility agents”), antifoam agents, cleaning agents, sequestering agents, drift reduction agents, neutralizing agents and buffers, corrosion inhibitors, dyes, odorants, spreading agents (also referred to as “spreaders”), penetration aids (also referred to as “penetrants”), sticking agents (also referred to as “stickers” or “binders”), dispersing agents, thickening agents (also referred to as “thickeners”), stabilizers, emulsifiers, freezing point depressants, antimicrobial agents, and the like.

Carriers

In some embodiments, a pheromone composition can include a carrier. The carrier can be, but is not limited to, an inert liquid or solid.

Examples of solid carriers include but are not limited to fillers such as kaolin, bentonite, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth, wax, gypsum, diatomaceous earth, rubber, plastic, China clay, mineral earths such as silicas, silica gels, silicates, attaclay, limestone, chalk, loess, clay, dolomite, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal meals, tree bark meal, wood meal and nutshell meal, cellulose powders, attapulgites, montmorillonites, mica, vermiculites, synthetic silicas and synthetic calcium silicates, or compositions of these.

Examples of liquid carriers include, but are not limited to, water; alcohols, such as ethanol, butanol or glycol, as well as their ethers or esters, such as methylglycol acetate; ketones, such as acetone, cyclohexanone, methylethyl ketone, methylisobutylketone, or isophorone; alkanes such as hexane, pentane, or heptanes; aromatic hydrocarbons, such as xylenes or alkyl naphthalenes; mineral or vegetable oils; aliphatic chlorinated hydrocarbons, such as trichloroethane or methylene chloride; aromatic chlorinated hydrocarbons, such as chlorobenzenes; water-soluble or strongly polar solvents such as dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone; liquefied gases; waxes, such as beeswax, lanolin, shellac wax, carnauba wax, fruit wax (such as bayberry or sugar cane wax) candelilla wax, other waxes such as microcrystalline, ozocerite, ceresin, or montan; salts such as monoethanolamine salt, sodium sulfate, potassium sulfate, sodium chloride, potassium chloride, sodium acetate, ammonium hydrogen sulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammonium carbamateand mixtures thereof. Baits or feeding stimulants can also be added to the carrier.

Synergist

In some embodiments, the pheromone composition is combined with an active chemical agent such that a synergistic effect results. The synergistic effect obtained by the taught methods can be quantified according to Colby's formula (i.e. (E)=X+Y−(X*Y/100). See Colby, R. S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations”, 1967 Weeds, vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, by “synergistic” is intended a component which, by virtue of its presence, increases the desired effect by more than an additive amount. The pheromone compositions and adjuvants of the present methods can synergistically increase the effectiveness of agricultural active compounds and also agricultural auxiliary compounds.

Thus, in some embodiments, a pheromone composition can be formulated with a synergist. The term, “synergist,” as used herein, refers to a substance that can be used with a pheromone for reducing the amount of the pheromone dose or enhancing the effectiveness of the pheromone for attracting at least one species of insect. The synergist may or may not be an independent attractant of an insect in the absence of a pheromone.

In some embodiments, the synergist is a volatile phytochemical that attracts at least one species of Lepidoptera. The term, “phytochemical,” as used herein, means a compound occurring naturally in a plant species. In a particular embodiment, the synergist is selected from the group comprising β-caryophyllene, iso-caryophyllene, α-humulene, inalool, Z3-hexenol/yl acetate, β-farnesene, benzaldehyde, phenylacetaldehyde, and combinations thereof.

The pheromone composition can contain the pheromone and the synergist in a mixed or otherwise combined form, or it may contain the pheromone and the synergist independently in a non-mixed form.

Insecticide

The pheromone composition can include one or more insecticides. In one embodiment, the insecticides are chemical insecticides known to one skilled in the art. Examples of the chemical insecticides include one or more of pyrethoroid or organophosphorus insecticides, including but are not limited to, cyfluthrin, permethrin, cypermethrin, bifinthrin, fenvalerate, flucythrinate, azinphosmethyl, methyl parathion, buprofezin, pyriproxyfen, flonicamid, acetamiprid, dinotefuran, clothianidin, acephate, malathion, quinolphos, chloropyriphos, profenophos, bendiocarb, bifenthrin, chlorpyrifos, cyfluthrin, diazinon, pyrethrum, fenpropathrin, kinoprene, insecticidal soap or oil, neonicotinoids, diamides, avermectin and derivatives, spinosad and derivatives, azadirachtin, pyridalyl, and mixtures thereof.

In another embodiment, the insecticides are one or more biological insecticides known to one skilled in the art. Examples of the biological insecticides include, but are not limited to, azadirachtin (neem oil), toxins from natural pyrethrins, Bacillus thuringiencis and Beauveria bassiana, viruses (e.g., CYD-X™, CYD-X HP™, Germstar™ Madex H P™ and Spod-X™), peptides (Spear-T™, Spear-P™, and Spear-C™)

In another embodiment, the insecticides are insecticides that target the nerve and muscle. Examples include acetylcholinesterase (AChE) inhibitors, such as carbamates (e.g., methomyl and thiodicarb) and organophosphates (e.g., chlorpyrifos) GABA-gated chloride channel antagonists, such as cyclodiene organochlorines (e.g., endosulfan) and phenylpyrazoles (e.g., fipronil), sodium channel modulators, such as pyrethrins and pyrethroids (e.g., cypermethrin and λ-cyhalothrin), nicotinic acetylcholine receptor (nAChR) agonists, such as neonicotinoids (e.g., acetamiprid, tiacloprid, thiamethoxam), nicotinic acetylcholine receptor (nAChR) allosteric modulators, such as spinosyns (e.g., spinose and spinetoram), chloride channel activators, such as avermectins and milbemycins (e.g., abamectin, emamectin benzoate), Nicotinic acetylcholine receptor (nAChR) blockers, such as bensultap and cartap, voltage dependent sodium channel blockers, such as indoxacarb and metaflumizone, ryanodine receptor modulator, such as diamides (e.g. dhlorantraniliprole and flubendiamide). In another embodiment, the insecticides are insecticides that target respiration. Examples include chemicals that uncouple oxidative phosphorylation via disruption of the proton gradient, such as chlorfenapyr, and mitochondrial complex I electron transport inhibitors.

In another embodiment, the insecticides are insecticides that target midgut. Examples include microbial disruptors of insect midgut membranes, such as Bacillus thuringiensis and Bacillus sphaericus.

In another embodiment, the insecticides are insecticides that target growth and development. Examples include juvenile hormone mimics, such as juvenile hormone analogues (e.g. fenoxycarb), inhibitors of chitin biosynthesis, Type 0, such as benzoylureas (e.g., flufenoxuron, lufenuron, and novaluron), and ecdysone receptor agonists, such as diacylhydrazines (e.g., methoxyfenozide and tebufenozide)

Stabilizer

According to another embodiment of the disclosure, the pheromone composition may include one or more additives that enhance the stability of the composition. Examples of additives include, but are not limited to, fatty acids and vegetable oils, such as for example olive oil, soybean oil, corn oil, safflower oil, canola oil, and combinations thereof.

Filler

According to another embodiment of the disclosure, the pheromone composition may include one or more fillers. Examples of fillers include, but are not limited to, one or more mineral clays (e.g., attapulgite). In some embodiments, the attractant-composition may include one or more organic thickeners. Examples of such thickeners include, but are not limited to, methyl cellulose, ethyl cellulose, and any combinations thereof.

Solvent

According to another embodiment, the pheromone compositions of the present disclosure can include one or more solvents. Compositions containing solvents are desirable when a user is to employ liquid compositions which may be applied by brushing, dipping, rolling, spraying, or otherwise applying the liquid compositions to substrates on which the user wishes to provide a pheromone coating (e.g., a lure). In some embodiments, the solvent(s) to be used is/are selected so as to solubilize, or substantially solubilize, the one or more ingredients of the pheromone composition. Examples of solvents include, but are not limited to, water, aqueous solvent (e.g., mixture of water and ethanol), ethanol, methanol, chlorinated hydrocarbons, petroleum solvents, turpentine, xylene, and any combinations thereof.

In some embodiments, the pheromone compositions of the present disclosure comprise organic solvents. Organic solvents are used mainly in the formulation of emulsifiable concentrates, ULV formulations, and to a lesser extent granular formulations. Sometimes mixtures of solvents are used. In some embodiments, the present disclosure teaches the use of solvents including aliphatic paraffinic oils such as kerosene or refined paraffins. In other embodiments, the present disclosure teaches the use of aromatic solvents such as xylene and higher molecular weight fractions of C9 and C10 aromatic solvents. In some embodiments, chlorinated hydrocarbons are useful as co-solvents to prevent crystallization when the formulation is emulsified into water. Alcohols are sometimes used as co-solvents to increase solvent power.

Solubilizing Agent

In some embodiments, the pheromone compositions of the present disclosure comprise solubilizing agents. A solubilizing agent is a surfactant, which will form micelles in water at concentrations above the critical micelle concentration. The micelles are then able to dissolve or solubilize water-insoluble materials inside the hydrophobic part of the micelle. The types of surfactants usually used for solubilization are non-ionics: sorbitan monooleates; sorbitan monooleate ethoxylates; and methyl oleate esters.

Binder

According to another embodiment of the disclosure, the pheromone composition may include one or more binders. Binders can be used to promote association of the pheromone composition with the surface of the material on which said composition is coated. In some embodiments, the binder can be used to promote association of another additive (e.g., insecticide, insect growth regulators, and the like) to the pheromone composition and/or the surface of a material. For example, a binder can include a synthetic or natural resin typically used in paints and coatings. These may be modified to cause the coated surface to be friable enough to allow insects to bite off and ingest the components of the composition (e.g., insecticide, insect growth regulators, and the like), while still maintaining the structural integrity of the coating.

Non-limiting examples of binders include polyvinylpyrrolidone, polyvinyl alcohol, partially hydrolyzed polyvinyl acetate, carboxymethylcellulose, starch, vinylpyrrolidone/vinyl acetate copolymers and polyvinyl acetate, or compositions of these; lubricants such as magnesium stearate, sodium stearate, talc or polyethylene glycol, or compositions of these; antifoams such as silicone emulsions, long-chain alcohols, phosphoric esters, acetylene diols, fatty acids or organofluorine compounds, and complexing agents such as: salts of ethylenediaminetetraacetic acid (EDTA), salts of trinitrilotriacetic acid or salts of polyphosphoric acids, or compositions of these.

In some embodiments, the binder also acts a filler and/or a thickener. Examples of such binders include, but are not limited to, one or more of shellac, acrylics, epoxies, alkyds, polyurethanes, linseed oil, tung oil, and any combinations thereof.

Surface-Active Agents

In some embodiments, the pheromone compositions comprise surface-active agents. In some embodiments, the surface-active agents are added to liquid agricultural compositions. In other embodiments, the surface-active agents are added to solid formulations, especially those designed to be diluted with a carrier before application. Thus, in some embodiments, the pheromone compositions comprise surfactants. Surfactants are sometimes used, either alone or with other additives, such as mineral or vegetable oils as adjuvants to spray-tank mixes to improve the biological performance of the pheromone on the target. The surface-active agents can be anionic, cationic, or nonionic in character, and can be employed as emulsifying agents, wetting agents, suspending agents, or for other purposes. In some embodiments, the surfactants are non-ionics such as: alky ethoxylates, linear aliphatic alcohol ethoxylates, and aliphatic amine ethoxylates. Surfactants conventionally used in the art of formulation and which may also be used in the present formulations are described, in McCutcheon's Detergents and Emulsifiers Annual, MC Publishing Corp., Ridgewood, N.J., 1998, and in Encyclopedia of Surfactants, Vol. I-III, Chemical Publishing Co., New York, 1980-81. In some embodiments, the present disclosure teaches the use of surfactants including alkali metal, alkaline earth metal or ammonium salts of aromatic sulfonic acids, for example, ligno-, phenol-, naphthalene- and dibutylnaphthalenesulfonic acid, and of fatty acids of arylsulfonates, of alkyl ethers, of lauryl ethers, of fatty alcohol sulfates and of fatty alcohol glycol ether sulfates, condensates of sulfonated naphthalene and its derivatives with formaldehyde, condensates of naphthalene or of the naphthalenesulfonic acids with phenol and formaldehyde, condensates of phenol or phenolsulfonic acid with formaldehyde, condensates of phenol with formaldehyde and sodium sulfite, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-, octyl- or nonylphenol, tributylphenyl polyglycol ether, alkylaryl polyether alcohols, isotridecyl alcohol, ethoxylated castor oil, ethoxylated triarylphenols, salts of phosphated triarylphenolethoxylates, lauryl alcohol polyglycol ether acetate, sorbitol esters, lignin-sulfite waste liquors or methylcellulose, or compositions of these.

In some embodiments, the present disclosure teaches other suitable surface-active agents, including salts of alkyl sulfates, such as diethanolammonium lauryl sulfate; alkylarylsulfonate salts, such as calcium dodecylbenzenesulfonate; alkylphenol-alkylene oxide addition products, such as nonylphenol-C₁₈ ethoxylate; alcohol-alkylene oxide addition products, such as tridecyl alcohol-C₁₆ ethoxylate; soaps, such as sodium stearate; alkylnaphthalene-sulfonate salts, such as sodium dibutyl-naphthalenesulfonate; dialkyl esters of sulfosuccinate salts, such as sodium di(2-ethylhexyl)sulfosuccinate; sorbitol esters, such as sorbitol oleate; quaternary amines, such as lauryl trimethylammonium chloride; polyethylene glycol esters of fatty acids, such as polyethylene glycol stearate; block copolymers of ethylene oxide and propylene oxide; salts of mono and dialkyl phosphate esters; vegetable oils such as soybean oil, rapeseed/canola oil, olive oil, castor oil, sunflower seed oil, coconut oil, corn oil, cottonseed oil, linseed oil, palm oil, peanut oil, safflower oil, sesame oil, tung oil and the like; and esters of the above vegetable oils, particularly methyl esters.

Wetting Agents

In some embodiments, the pheromone compositions comprise wetting agents. A wetting agent is a substance that when added to a liquid increases the spreading or penetration power of the liquid by reducing the interfacial tension between the liquid and the surface on which it is spreading. Wetting agents are used for two main functions in agrochemical formulations: during processing and manufacture to increase the rate of wetting of powders in water to make concentrates for soluble liquids or suspension concentrates; and during mixing of a product with water in a spray tank or other vessel to reduce the wetting time of wettable powders and to improve the penetration of water into water-dispersible granules. In some embodiments, examples of wetting agents used in the pheromone compositions of the present disclosure, including wettable powders, suspension concentrates, and water-dispersible granule formulations are: sodium lauryl sulphate; sodium dioctyl sulphosuccinate; alkyl phenol ethoxylates; and aliphatic alcohol ethoxylates.

Dispersing Agent

In some embodiments, the pheromone compositions of the present disclosure comprise dispersing agents. A dispersing agent is a substance which adsorbs onto the surface of particles and helps to preserve the state of dispersion of the particles and prevents them from reaggregating. In some embodiments, dispersing agents are added to pheromone compositions of the present disclosure to facilitate dispersion and suspension during manufacture, and to ensure the particles redisperse into water in a spray tank. In some embodiments, dispersing agents are used in wettable powders, suspension concentrates, and water-dispersible granules. Surfactants that are used as dispersing agents have the ability to adsorb strongly onto a particle surface and provide a charged or steric barrier to re-aggregation of particles. In some embodiments, the most commonly used surfactants are anionic, non-ionic, or mixtures of the two types.

In some embodiments, for wettable powder formulations, the most common dispersing agents are sodium lignosulphonates. In some embodiments, suspension concentrates provide very good adsorption and stabilization using polyelectrolytes, such as sodium naphthalene sulphonate formaldehyde condensates. In some embodiments, tristyrylphenol ethoxylated phosphate esters are also used. In some embodiments, such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersing agents for suspension concentrates.

Polymeric Surfactant

In some embodiments, the pheromone compositions of the present disclosure comprise polymeric surfactants. In some embodiments, the polymeric surfactants have very long hydrophobic ‘backbones’ and a large number of ethylene oxide chains forming the ‘teeth’ of a ‘comb’ surfactant. In some embodiments, these high molecular weight polymers can give very good long-term stability to suspension concentrates, because the hydrophobic backbones have many anchoring points onto the particle surfaces. In some embodiments, examples of dispersing agents used in pheromone compositions of the present disclosure are: sodium lignosulphonates; sodium naphthalene sulphonate formaldehyde condensates; tristyrylphenol ethoxylate phosphate esters; aliphatic alcohol ethoxylates; alky ethoxylates; EO-PO block copolymers; and graft copolymers.

Emulsifying Agent

In some embodiments, the pheromone compositions of the present disclosure comprise emulsifying agents. An emulsifying agent is a substance, which stabilizes a suspension of droplets of one liquid phase in another liquid phase. Without the emulsifying agent the two liquids would separate into two immiscible liquid phases. In some embodiments, the most commonly used emulsifier blends include alkylphenol or aliphatic alcohol with 12 or more ethylene oxide units and the oil-soluble calcium salt of dodecylbenzene sulphonic acid. A range of hydrophile-lipophile balance (“HLB”) values from 8 to 18 will normally provide good stable emulsions. In some embodiments, emulsion stability can sometimes be improved by the addition of a small amount of an EO-PO block copolymer surfactant.

Gelling Agent

In some embodiments, the pheromone compositions comprise gelling agents. Thickeners or gelling agents are used mainly in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheology or flow properties of the liquid and to prevent separation and settling of the dispersed particles or droplets. Thickening, gelling, and anti-settling agents generally fall into two categories, namely water-insoluble particulates and water-soluble polymers. It is possible to produce suspension concentrate formulations using clays and silicas. In some embodiments, the pheromone compositions comprise one or more thickeners including, but not limited to: montmorillonite, e.g. bentonite; magnesium aluminum silicate; and attapulgite. In some embodiments, the present disclosure teaches the use of polysaccharides as thickening agents. The types of polysaccharides most commonly used are natural extracts of seeds and seaweeds or synthetic derivatives of cellulose. Some embodiments utilize xanthan and some embodiments utilize cellulose. In some embodiments, the present disclosure teaches the use of thickening agents including, but are not limited to: guar gum; locust bean gum; carrageenam; alginates; methyl cellulose; sodium carboxymethyl cellulose (SCMC); hydroxyethyl cellulose (HEC). In some embodiments, the present disclosure teaches the use of other types of anti-settling agents such as modified starches, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

Anti-Foam Agent

In some embodiments, the presence of surfactants, which lower interfacial tension, can cause water-based formulations to foam during mixing operations in production and in application through a spray tank. Thus, in some embodiments, in order to reduce the tendency to foam, anti-foam agents are often added either during the production stage or before filling into bottles/spray tanks. Generally, there are two types of anti-foam agents, namely silicones and nonsilicones. Silicones are usually aqueous emulsions of dimethyl polysiloxane, while the nonsilicone anti-foam agents are water-insoluble oils, such as octanol and nonanol, or silica. In both cases, the function of the anti-foam agent is to displace the surfactant from the air-water interface.

Preservative

In some embodiments, the pheromone compositions comprise a preservative.

Additional Active Agent

According to another embodiment of the disclosure, the pheromone composition may include one or more insect feeding stimulants. Examples of insect feeding stimulants include, but are not limited to, crude cottonseed oil, fatty acid esters of phytol, fatty acid esters of geranyl geraniol, fatty acid esters of other plant alcohols, plant extracts, and combinations thereof.

According to another embodiment of the disclosure, the pheromone composition may include one or more insect growth regulators (“IGRs”). IGRs may be used to alter the growth of the insect and produce deformed insects. Examples of insect growth regulators include, for example, dimilin.

According to another embodiment of the disclosure, the attractant-composition may include one or more insect sterilants that sterilize the trapped insects or otherwise block their reproductive capacity, thereby reducing the population in the following generation. In some situations allowing the sterilized insects to survive and compete with non-trapped insects for mates is more effective than killing them outright.

Sprayable Compositions

In some embodiments, the pheromone compositions disclosed herein can be formulated as a sprayable composition (i.e., a sprayable pheromone composition). An aqueous solvent can be used in the sprayable composition, e.g., water or a mixture of water and an alcohol, glycol, ketone, or other water-miscible solvent. In some embodiments, the water content of such mixture is at least about 10%, at least about 20%, at least about 30%, at least about 40%, 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some embodiments, the sprayable composition is concentrate, i.e. a concentrated suspension of the pheromone, and other additives (e.g., a waxy substance, a stabilizer, and the like) in the aqueous solvent, and can be diluted to the final use concentration by addition of solvent (e.g., water).

In some embodiments, the a waxy substance can be used as a carrier for the pheromone and its positional isomer in the sprayable composition. The waxy substance can be, e.g., a biodegradable wax, such as bees wax, carnauba wax and the like, candelilla wax (hydrocarbon wax), montan wax, shellac and similar waxes, saturated or unsaturated fatty acids, such as lauric, palmitic, oleic or stearic acid, fatty acid amides and esters, hydroxylic fatty acid esters, such as hydroxyethyl or hydroxypropyl fatty acid esters, fatty alcohols, and low molecular weight polyesters such as polyalkylene succinates.

In some embodiments, a stabilizer can be used with the sprayable pheromone compositions. The stabilizer can be used to regulate the particle size of concentrate and/or to allow the preparation of a stable suspension of the pheromone composition. In some embodiments, the stabilizer is selected from hydroxylic and/or ethoxylated polymers. Examples include ethylene oxide and propylene oxide copolymer, polyalcohols, including starch, maltodextrin and other soluble carbohydrates or their ethers or esters, cellulose ethers, gelatin, polyacrylic acid and salts and partial esters thereof and the like. In other embodiments, the stabilizer can include polyvinyl alcohols and copolymers thereof, such as partly hydrolyzed polyvinyl acetate. The stabilizer may be used at a level sufficient to regulate particle size and/or to prepare a stable suspension, e.g., between 0.1% and 15% of the aqueous solution.

In some embodiments, a binder can be used with the sprayable pheromone compositions. In some embodiments, the binder can act to further stabilize the dispersion and/or improve the adhesion of the sprayed dispersion to the target locus (e.g., trap, lure, plant, and the like). The binder can be polysaccharide, such as an alginate, cellulose derivative (acetate, alkyl, carboxymethyl, hydroxyalkyl), starch or starch derivative, dextrin, gum (arabic, guar, locust bean, tragacanth, carrageenan, and the like), sucrose, and the like. The binder can also be a non-carbohydrate, water-soluble polymer such as polyvinyl pyrrolidone, or an acidic polymer such as polyacrylic acid or polymethacrylic acid, in acid and/or salt form, or mixtures of such polymers.

Microencapsulated Pheromones

In some embodiments, the pheromone compositions disclosed herein can be formulated as a microencapsulated pheromone, such as disclosed in Ill'lchev, A L et al., J. Econ. Entomol. 2006; 99(6):2048-54; and Stelinki, L L et al., J. Econ. Entomol. 2007; 100(4):1360-9. Microencapsulated pheromones (MECs) are small droplets of pheromone enclosed within polymer capsules. The capsules control the release rate of the pheromone into the surrounding environment, and are small enough to be applied in the same method as used to spray insecticides. The effective field longevity of the microencapsulated pheromone formulations can range from a few days to slightly more than a week, depending on inter alia climatic conditions, capsule size and chemical properties.

Slow-Release Formulation

Pheromone compositions can be formulated so as to provide slow release into the atmosphere, and/or so as to be protected from degradation following release. For example, the pheromone compositions can be included in carriers such as microcapsules, biodegradable flakes and paraffin wax-based matrices. Alternatively, the pheromone composition can be formulated as a slow release sprayable.

In certain embodiments, the pheromone composition may include one or more polymeric agents known to one skilled in the art. The polymeric agents may control the rate of release of the composition to the environment. In some embodiments, the polymeric attractant-composition is impervious to environmental conditions. The polymeric agent may also be a sustained-release agent that enables the composition to be released to the environment in a sustained manner.

Examples of polymeric agents include, but are not limited to, celluloses, proteins such as casein, fluorocarbon-based polymers, hydrogenated rosins, lignins, melamine, polyurethanes, vinyl polymers such as polyvinyl acetate (PVAC), polycarbonates, polyvinylidene dinitrile, polyamides, polyvinyl alcohol (PVA), polyamide-aldehyde, polyvinyl aldehyde, polyesters, polyvinyl chloride (PVC), polyethylenes, polystyrenes, polyvinylidene, silicones, and combinations thereof. Examples of celluloses include, but are not limited to, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate-butyrate, cellulose acetate-propionate, cellulose propionate, and combinations thereof.

Other agents which can be used in slow-release or sustained-release formulations include fatty acid esters (such as a sebacate, laurate, palmitate, stearate or arachidate ester) or a fatty alcohols (such as undecanol, dodecanol, tridecanol, tridecenol, tetradecanol, tetradecenol, tetradecadienol, pentadecanol, pentadecenol, hexadecanol, hexadecenol, hexadecadienol, octadecenol and octadecadienol).

Administration of Pheromone Composition Lures

The pheromone compositions of the present disclosure may be coated on or sprayed on a lure, or the lure may be otherwise impregnated with a pheromone composition.

Traps

The pheromone compositions of the disclosure may be used in traps, such as those commonly used to attract any insect species, e.g., insects of the order Lepidoptera. Such traps are well known to one skilled in the art, and are commonly used in many states and countries in insect eradication programs. In one embodiment, the trap includes one or more septa, containers, or storage receptacles for holding the pheromone composition. Thus, in some embodiments, the present disclosure provides a trap loaded with at least one pheromone composition. Thus, the pheromone compositions of the present disclosure can be used in traps for example to attract insects as part of a strategy for insect monitoring, mass trapping, mating disruption, or lure/attract and kill for example by incorporating a toxic substance into the trap to kill insects caught.

Mass trapping involves placing a high density of traps in a crop to be protected so that a high proportion of the insects are removed before the crop is damaged. Lure/attract-and-kill techniques are similar except once the insect is attracted to a lure, it is subjected to a killing agent. Where the killing agent is an insecticide, a dispenser can also contain a bait or feeding stimulant that will entice the insects to ingest an effective amount of an insecticide. The insecticide may be an insecticide known to one skilled in the art. The insecticide may be mixed with the attractant-composition or may be separately present in a trap. Mixtures may perform the dual function of attracting and killing the insect.

Such traps may take any suitable form, and killing traps need not necessarily incorporate toxic substances, the insects being optionally killed by other means, such as drowning or electrocution. Alternatively, the traps can contaminate the insect with a fungus or virus that kills the insect later. Even where the insects are not killed, the trap can serve to remove the male insects from the locale of the female insects, to prevent breeding.

It will be appreciated by a person skilled in the art that a variety of different traps are possible. Suitable examples of such traps include water traps, sticky traps, and one-way traps. Sticky traps come in many varieties. One example of a sticky trap is of cardboard construction, triangular or wedge-shaped in cross-section, where the interior surfaces are coated with a non-drying sticky substance. The insects contact the sticky surface and are caught. Water traps include pans of water and detergent that are used to trap insects. The detergent destroys the surface tension of the water, causing insects that are attracted to the pan, to drown in the water. One-way traps allow an insect to enter the trap but prevent it from exiting. The traps of the disclosure can be colored brightly, to provide additional attraction for the insects.

In some embodiments, the pheromone traps containing the composition may be combined with other kinds of trapping mechanisms. For example, in addition to the pheromone composition, the trap may include one or more florescent lights, one or more sticky substrates and/or one or more colored surfaces for attracting moths. In other embodiments, the pheromone trap containing the composition may not have other kinds of trapping mechanisms.

The trap may be set at any time of the year in a field. Those of skill in the art can readily determine an appropriate amount of the compositions to use in a particular trap, and can also determine an appropriate density of traps/acre of crop field to be protected.

The trap can be positioned in an area infested (or potentially infested) with insects. Generally, the trap is placed on or close to a tree or plant. The aroma of the pheromone attracts the insects to the trap. The insects can then be caught, immobilized and/or killed within the trap, for example, by the killing agent present in the trap.

Traps may also be placed within an orchard to overwhelm the pheromones emitted by the females, so that the males simply cannot locate the females. In this respect, a trap need be nothing more than a simple apparatus, for example, a protected wickable to dispense pheromone.

The traps of the present disclosure may be provided in made-up form, where the compound of the disclosure has already been applied. In such an instance, depending on the half-life of the compound, the compound may be exposed, or may be sealed in conventional manner, such as is standard with other aromatic dispensers, the seal only being removed once the trap is in place.

Alternatively, the traps may be sold separately, and the compound of the disclosure provided in dispensable format so that an amount may be applied to trap, once the trap is in place. Thus, the present disclosure may provide the compound in a sachet or other dispenser.

Dispenser

Pheromone compositions can be used in conjunction with a dispenser for release of the composition in a particular environment. Any suitable dispenser known in the art can be used. Examples of such dispensers include but are not limited to, aerosol emitters, hand-applied dispensers, bubble caps comprising a reservoir with a permeable barrier through which pheromones are slowly released, pads, beads, tubes rods, spirals or balls composed of rubber, plastic, leather, cotton, cotton wool, wood or wood products that are impregnated with the pheromone composition. For example, polyvinyl chloride laminates, pellets, granules, ropes or spirals from which the pheromone composition evaporates, or rubber septa. One of skill in the art will be able to select suitable carriers and/or dispensers for the desired mode of application, storage, transport or handling.

In another embodiment, a device may be used that contaminates the male insects with a powder containing the pheromone substance itself. The contaminated males then fly off and provide a source of mating disruption by permeating the atmosphere with the pheromone substance, or by attracting other males to the contaminated males, rather than to real females.

Behavior Modification

Pheromone compositions prepared according to the methods disclosed herein can be used to control or modulate the behavior of insects. In some embodiments, the behavior of the target insect can be modulated in a tunable manner inter alia by varying the ratio of the pheromone to the positional isomer in the composition such that the insect is attracted to a particular locus but does not contact said locus or such the insect in fact contacts said locus. Thus, in some embodiments, the pheromones can be used to attract insects away from vulnerable crop areas. Accordingly, the disclosure also provides a method for attracting insects to a locus. The method includes administering to a the locus an effective amount of the pheromone composition.

The method of mating disruption may include periodically monitoring the total number or quantity of the trapped insects. The monitoring may be performed by counting the number of insects trapped for a predetermined period of time such as, for example, daily, Weekly, bi-Weekly, monthly, once-in-three months, or any other time periods selected by the monitor. Such monitoring of the trapped insects may help estimate the population of insects for that particular period, and thereby help determine a particular type and/or dosage of pest control in an integrated pest management system. For example, a discovery of a high insect population can necessitate the use of methods for removal of the insect. Early warning of an infestation in a new habitat can allow action to be taken before the population becomes unmanageable. Conversely, a discovery of a low insect population can lead to a decision that it is sufficient to continue monitoring the population. Insect populations can be monitored regularly so that the insects are only controlled when they reach a certain threshold. This provides cost-effective control of the insects and reduces the environmental impact of the use of insecticides.

Mating Disruption

Pheromones prepared according to the methods of the disclosure can also be used to disrupt mating. Mating disruption is a pest management technique designed to control insect pests by introducing artificial stimuli (e.g., a pheromone composition as disclosed herein) that confuses the insects and disrupts mating localization and/or courtship, thereby preventing mating and blocking the reproductive cycle.

In many insect species of interest to agriculture, such as those in the order Lepidoptera, females emit an airborne trail of a specific chemical blend constituting that species' sex pheromone. This aerial trail is referred to as a pheromone plume. Males of that species use the information contained in the pheromone plume to locate the emitting female (known as a “calling” female). Mating disruption exploits the male insects' natural response to follow the plume by introducing a synthetic pheromone into the insects' habitat, which is designed to mimic the sex pheromone produced by the female insect. Thus, in some embodiments, the synthetic pheromone utilized in mating disruption is a synthetically derived pheromone composition comprising a pheromone having a chemical structure of a sex pheromone and a positional isomer thereof which is not produced by the target insect.

The general effect of mating disruption is to confuse the male insects by masking the natural pheromone plumes, causing the males to follow “false pheromone trails” at the expense of finding mates, and affecting the males' ability to respond to “calling” females. Consequently, the male population experiences a reduced probability of successfully locating and mating with females, which leads to the eventual cessation of breeding and collapse of the insect infestation

Strategies of mating disruption include confusion, trail-masking and false-trail following. Constant exposure of insects to a high concentration of a pheromone can prevent male insects from responding to normal levels of the pheromone released by female insects. Trail-masking uses a pheromone to destroy the trail of pheromones released by females. False-trail following is carried out by laying numerous spots of a pheromone in high concentration to present the male with many false trails to follow. When released in sufficiently high quantities, the male insects are unable to find the natural source of the sex pheromones (the female insects) so that mating cannot occur.

In some embodiments, a wick or trap may be adapted to emit a pheromone for a period at least equivalent to the breeding season(s) of the midge, thus causing mating disruption. If the midge has an extended breeding season, or repeated breeding season, the present disclosure provides a wick or trap capable of emitting pheromone for a period of time, especially about two weeks, and generally between about 1 and 4 weeks and up to 6 weeks, which may be rotated or replaced by subsequent similar traps. A plurality of traps containing the pheromone composition may be placed in a locus, e.g., adjacent to a crop field. The locations of the traps, and the height of the traps from ground may be selected in accordance with methods known to one skilled in the art.

Alternatively, the pheromone composition may be dispensed from formulations such as microcapsules or twist-ties, such as are commonly used for disruption of the mating of insect pests.

A variety of pheromones, including those set forth in Table 1 can be prepared according to the methods and formulations as described above. For example, the methods can be used to synthesize a corn earworm (H. zea) sex pheromone blend, which is generally understood in the art to entail a mixture of (Z)-hexadeca-9-en-1-al (3%) and (Z)-hexadeca-11-en-1-al (97%). However, as disclosed herein, the pheromone blend can be doped with (Z)-hexadeca-5-en-1-al to tunably elicit a response in the male corn earworms. Thus, the corn earworm sex pheromone can be used in conjunction with a sustained pheromone release device having a polymer container containing a mixture of the sex pheromone and a fatty acid ester (such as a sebacate, laurate, palmitate, stearate or arachidate ester) or a fatty alcohol (such as undecanol, dodecanol, tridecanol, tridecenol, tetradecanol, tetradecenol, tetradecadienol, pentadecanol, pentadecenol, hexadecanol, hexadecenol, hexadecadienol, octadecenol and octadecadienol). The polymer container can be a tube, an ampule, or a bag made of a polyolefin or an olefin component-containing copolymer. Sex pheromones of other pest insects, such as, but not limited to, the cotton bollworm (Helicoverpa armigera), fall army worm (Spodoptera frugiperda), oriental fruit moth (Grapholita molesta), peach twig borer (Anarsia lineatella), diamondback moth (Plutella xylostella), soybean looper (Chrysodeixis includes) and leaf roller (Tortricidae) can be used in this type of sustained pheromone release device.

As will be apparent to one of skill in the art, the amount of a pheromone or pheromone composition used for a particular application can vary depending on several factors such as the type and level of insect infestation; the type of composition used; the concentration of the active components; how the composition is provided, for example, the type of dispenser used; the type of location to be treated; the length of time the method is to be used for; and environmental factors such as temperature, wind speed and direction, rainfall and humidity. Those of skill in the art will be able to determine an effective amount of a pheromone or pheromone composition for use in a given application.

EXAMPLES

The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the disclosure in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.

Example 1: Hydroxylation of (Z)-5-Hexadecene by Cytochromes P450 of the CYP52 Family

The purpose of this example is to illustrate the biocatalytic hydroxylation of (Z)-5-hexadecene by members of the CYP52 family.

Two P450 cytochromes of the CYP52 family were integrated into the P. pastoris CBS7435 Mut^(s) genome along with their corresponding cytochrome P450 reductases (CPR). Biotransformations were performed with these strains to determine whether these P450s hydroxylate (Z)-5-hexadecene. Strains and oligonucleotides disclosed in this example are listed in Tables 11 and 12.

TABLE 11 Genotypes of strains used in Example 1. Strain No. Genotype SPV048 P. pastoris CBS7435 mut^(S) pPpT4_SmiI_cmRED_cmCYP52A3 SPV051 P. pastoris CBS7435 mut^(S) pPpT4_SmiI_ctRED_ctCYP52A13

TABLE 12 Oligonucleotide sequences used in Example 1. SEQ ID Primer Sequence Description NO. OPV ATGACGGTTCATGACATCATCGC CYP52A13 1 0042 forward primer OPV CTGACATCCTCTTGAGCGGC CYP52A13/ 2 0043 A3 reverse primer OPV ATGGCTATTGAGCAGATTATCGAAG CYP52A3 3 0044 forward primer

Gene sequences for C. tropicalis CYP52A13 (Accession No. AA073953.1), C. tropicalis CPR (Accession No. P37201.1), C. maltosa CYP52A3 (Accession No. P24458.1), as well as the C. maltosa CPR (Accession No. P50126.1), were ordered as synthetic genes (DNA 2.0, Menlo Park, Calif., USA), and cloned into the pT4_S vector using EcoRI/NotI restriction sites for directional cloning. The plasmid containing the expression cassettes for CYP52A3/CPR and CYP52A13/CPR under the control of an AOX promoter and terminator were linearized using the restriction enzyme SmiI and purified. Next, 500 ng of the linearized DNA sequences for expressing CYP52A3/CPR (SEQ ID NO:4) and CYP52A13/CPR (SEQ ID NO:5) were used to transform P. pastoris CBS7435 Mut^(s). The parent strain and the generation of the pT4 S plasmid used to generate the subsequent constructs are described by Gudiminchi et al. (Biotechnology Journal, 2013, 8(1), 146-52).

Colony PCR of the obtained P. pastoris strains was performed to verify the P450 enzymes CYP52A3 and CYP52A13 were present using the Failsafe™ PCR Kit (EPICENTRE® Biotechnologies, Madison, Wis.; Catalog #FS99060) using Premix D and primers shown in Table 21 according to the manufactures recommendations.

Shake flask cultivations of the strains SPV048 and SPV051 were started from single colonies derived from an YBD agar plate (10 g/L Bacto™ yeast extract, 20 g/L Bacto™ peptone, 20 g/L D (+) glucose, 15 g/L agar) containing 100 mg/L Zeocin™. A volume of 45 mL of BMD1 medium (BMD1(1 L): 10 g/L D (+) glucose autoclaved, 200 mL 10×PPB (10×PPB: 30.0 g/L K₂HPO₄, 118 g/L KH₂PO₄, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500×buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H₂O to 1 L) was inoculated with a single colony and incubated for approximately 63 h at 28° C. to 30° C. and 130 rpm in a 250 mL baffled Erlenmeyer flask. After the initial 63 h incubation 5 mL of BMM10 medium (BMM10 (1 L): 50 mL methanol, 200 mL 10×PPB (10×PPB: 30.0 g/L K₂HPO₄, 118 g/L KH₂PO₄, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500×buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H₂O to 1 L) was added. The cultivations were incubated for 12 h at 28° C. to 30° C., 130 rpm. After 12 hours incubation 0.4 mL of methanol was added to induce expression of the P450 enzymes and their corresponding CPR's and incubated for 12 h at 28° C. to 30° C., 130 rpm. Thereafter, 0.4 mL of methanol was added every 12 h and incubated at 28° C. to 30° C., 130 rpm. Cells were harvested after induction for approximately 72 h to 80 h and a total cultivation time of approximately 132 h to 143 h.

As control a volume of 45 mL of BMD1 medium (BMD1 (1 L): 10 g/L D (+) glucose autoclaved, 200 mL 10×PPB (10×PPB: 30.0 g/L K₂HPO₄, 118 g/L KH₂PO₄, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500×buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H₂O to 1 L) was inoculated with a single colony of strain SPV051 incubated for approximately 63 h at 28° C. to 30° C. and 130 rpm in a 250 mL baffled Erlenmeyer flask. After the initial 63 h incubation 5 mL of BMM10 medium without methanol (BMM10 without methanol (1 L): 200 mL 10×PPB (10×PPB: 30.0 g/L K₂HPO₄, 118 g/L KH₂PO₄, pH 6.0, autoclaved), 100 mL 10×YNB (10×YNB: 134 g/L Difco™ yeast nitrogen base without amino acids, autoclaved), 2 mL 500×buffer B (buffer B:10 mg/50 mL d-Biotin, filter sterilized), add autoclaved H₂O to 1 L) was added. The cultivations were incubated for additional 60 h to 68 h at 28° C. to 30° C., 130 rpm. Cells were harvested after a total cultivation time of approximately 132 h to 143 h.

Cultivations were harvested in 50 mL Falcon tubes via centrifugation at 3000×rcf for 5 min at 4° C. The supernatant was discarded. The pellet was resuspended in 5 mL 100 mM PPB (mix stock solutions: 80.2 mL of 1M K₂HPO₄ (174.18 g/L) with 19.8 mL of 1M KH₂PO₄ (136.09 g/L) autoclaved, add autoclaved H₂O to 1 L and adjust pH 7.4), containing 20% glycerol, pH 7.4 and centrifuged again at 3000×rcf for 5 min at 4° C. (washing step). The supernatant was discarded and the Falcon tube was carefully patted on a Kimwipe to remove excess buffer. Each pellet was weighed to determine the cell wet weight (cww) of the cultures. The washed pellet was resuspended in bioconversion buffer (100 mM PPB (mix stock solutions: 80.2 mL of 1M K₂HPO₄ (174.18 g/L) with 19.8 mL of 1M KH₂PO₄ (136.09 g/L) autoclaved, add autoclaved H₂O to 1 L and adjust to pH 7.4), 20% glycerol, 0.2% Emulgen 913 (Kao Chemicals, Japan), pH 7.4) targeting a final cell density of ˜200 g cww/L.

1 ml of the resuspended cultivation (200 g cww/L) was dispensed in a 50 mL Falcon tube. 125 μL neat substrate was added to each culture to initiate the bioconversion reactions. The bioconversion reactions were incubated at 30° C. and 200 rpm for 40 h to 48 h. The samples were stored at −80° C. until extraction and analysis of the respective product formation.

250 μL of 3 M HCl was added to each of the frozen samples. After addition of HCl samples were extracted twice with 1×1 mL or 2×2 mL diethyl ether. 10 μL of 10 mg/mL 1-Heptanol or 10 μL of 10 mg/mL 1-Tetradecanol was added to the sample as internal standard. Upon addition of diethyl ether and internal standard the sample was vortexed for 5 min. The entire sample was transferred to new reaction tubes and centrifuged for 10 min/8000×rcf at room temperature. The organic upper phase was transferred to a glass vial and air dried. The sample was resuspended to a final volume of 100 μL to 150 μL using Methyl Tertiary Butyl Ether (MTBE) or resuspended to a final volume of 200 μL using Tetrahydrofuran (THF) and analyzed via gas chromatography (GC).

An Agilent 6890 equipped with an FID detector and a J&W DB-23 column (length: 30 m, I.D. 25 mm, film 25 μm) was used to analyze the samples using the following program: Split ratio of 1:10. 240° C. for the injector inlet: 240° C. for the detector. H₂ at 40.0 mL/min, Air at 450 mL/min, Makeup flow (He) at 45 mL/min. Carrier He at 1.1 ml/min and 13 psi. 45° C. oven for 0.5 min; 5° C./min gradient to 50° C. then hold at 50° C. for 0.5 min.; 30° C./min gradient to 220° C., then hold at 220° C. for 3.33 min. Analysis was performed in triplicate using authentic standards (obtained from Sigma-Aldrich or Bedoukian Research).

Results are shown in Table 13 and FIG. 2. Surprisingly, the CYP52 enzymes exhibit selectivity for one end of the (Z)-5-hexadecene substrate over the other: The SPV048 bioconversion produced 66.8% (Z)-5-hexadecen-1-ol and 33.2% (Z)-11-hexadecen-1-ol while the SPV051 bioconversion produced 27.6% (Z)-5-hexadecen-1-ol and 74.4% (Z)-11-hexadecen-1-ol.

TABLE 13 Results for bioconversions. Induced Selectivity Strain Enzyme (Y/N) Substrate Products [%] SPV048 CYP52A3 N (Z)-5-hexadecene n.d. n/a SPV048 CYP52A3 Y (Z)-5-hexadecene (Z)-5-hexadecen-1-ol 66.8 ± 7.6 (Z)-11-hexadecen-1-ol 33.2 ± 1.0 SPV051 CYP52A13 N (Z)-5-hexadecene n.d. n/a SPV051 CYP52A13 Y (Z)-5-hexadecene (Z)-5-hexadecen-1-ol, 27.6 ± 4.3 (Z)-11-hexadecen-1-ol 74.4 ± 2.2

The results indicate that the biohydroxylation catalyst can functionalize an unsaturated hydrocarbon substrate on different termini to generate a mixture which includes a pheromone having a chemical structure corresponding to that of a natural insect pheromone produced by a given target member of the order Lepidoptera and a positional isomer of said sex pheromone, which is not naturally produced by said target insect.

Example 2. Synthesis of (Z)-11-Hexadecenol Carried Out According to Scheme 12

Z-5-hexadecene:

The cross metathesis reactions of 1-hexene and dodec-1-ene is carried out in a 250 mL three-necked round-bottomed flask fitted with a condenser, thermometer and septum. The dodec-1-ene (20 mL) is transferred to the reaction flask along with 4 mole equivalent of 1-hexene and the mixture is heated to the desired reaction temperature (ranging from 30 to 100° C.) using an oil bath on a controlled hotplate magnetic stirrer. Thereafter 0.5 mol % of the catalyst is added to the flask and the reaction mixture is continuously stirred with a magnetic stirrer bar until the formation of the primary metathesis products is completed. The progress of the reaction is monitored by GC/FID. The sample is prepared for GC analysis by diluting an aliquot (0.3 mL) of the sample, taken at various reaction time intervals, with 0.3 mL toluene and quenched with 2 drops of tert-butyl hydrogen peroxide prior to analysis. Once dodec-1-ene is completely consumed, the reaction is quenched with tert-butyl hydrogen peroxide and filtered through a plug of silica using hexane as eluent. The hexane filtrate is concentrated and the Z-5-hexadecene is isolated by distillation.

Z-11-Hexadecen-1-ol:

Z-5-Hexadecene is subjected to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent, concentrate and silica-gel chromatography.

Example 3. Synthesis of (Z)-11-Hexadecenol Carried Out According to Scheme 13

1-Dodecyne:

The synthesis of 1-dodecyne is carried out according to the protocol described in Oprean, Joan et al. Studia Universitatis Babes-Bolyai, Chemia, 2006, 51, 33.

5-Hexadecyne:

To a −78° C. solution of 1-dodecyne (5 mmol) in THF (20 mL), 2.5M n-BuLi (5 mmol) in hexane is added dropwise via a syringe. A solution of 1-bromobutane (5 mmol) and TBAI (0.2 mmol) dissolve in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH₄Cl and extract with hexanes (3×). The organic fractions are combined, dry with anhydrous MgSO₄ and concentrate under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1/hexane:ethyl acetate as mobile phase. Fractions containing the desired product are pulled and concentrate. 5-Hexadecyne is further purified by distillation.

Z-5-Hexadecene:

With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of Z-5-hexadecene (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford the desired product, Z-5-hexadecene, which can be further purified by distillation.

Z-11-Hexadecen-1-ol:

Z-5-hexadecene is then subject to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with ethyl acetate and further purified by distillation.

Example 4. Synthesis of (Z)-11-Hexadecenol Carried Out According to Scheme 14

1-Hexyne:

The synthesis of 1-hexyne is carried out according to the protocol described in Oprean, Joan et al. Studia Universitatis Babes-Bolyai, Chemia, 2006, 51, 33.

5-Hexadecyne:

To a −78° C. solution of 1-hexyne (5 mmol) in THF (20 mL), 2.5M n-BuLi (5 mmol) in hexane is added dropwise via a syringe. A solution of 1-bromodecane (5 mmol) and n-Bu₄NI (TBAI) (0.2 mmol) dissolve in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH₄Cl and extract with hexanes (3×). The organic fractions are combined, dry with anhydrous MgSO₄ and concentrate under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1/hexane:ethyl acetate as mobile phase. Fractions containing the desired product are pulled and concentrate.

Z-5-Hexadecene:

With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of Z-5-hexadecene (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford Z-5-hexadecene, which can be further purified by distillation.

Z-11-Hexadecen-1-ol:

Z-5-Hexadecene is then subjected to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and further purified by distillation.

Example 5. Synthesis of (Z)-11-Hexadecenol Carried Out According to Scheme 15

5-Hexadecyne:

To a −78° C. solution of 1-hexyne (0.383 g, 4.67 mmol) in THF (20 mL), 2.5 M n-BuLi (1.87 mL, 4.67 mmol) in hexane is added dropwise via a syringe. A solution of 1-bromodecane (4.67 mmol) and n-Bu₄NI (TBAI, 57 mg, 0.16 mmol) dissolved in THF is then dropwise added to the reaction mixture. The reaction mixture is allowed to warm to room temperature and then heat at 70° C. for 24 hours. The reaction is quenched with 5 mL of 1M NH₄Cl and extract with hexanes (3×). The organic fractions are combined, dried with anhydrous MgSO₄, and concentrated under reduced pressure. The resulting residue is purified by silica gel flash chromatography using 60:1 hexane:ethyl acetate as the mobile phase. Fractions containing the desired product, 5-hexadecyne, are pooled and concentrated.

Z-5-Hexadecene:

With stirring, a mixture of Lindlar's catalyst (40 mg) in pentane (10 mL) is put under a balloon of hydrogen for 90 min at 0° C. Quinoline (1 mg) is then added and the mixture is allowed to stir at 0° C. for another 30 min. A solution of Z-5-hexadecene (55 mg) in 2 mL of pentane is then added to the reaction mixture via a syringe. The reaction is allowed to warm to room temperature and the progress of the reaction is monitored by GC. After 18 hours of reaction time, the reaction mixture is filtered through a No. 4 Whatman filter paper and the filtrate is concentrated under reduced pressure to afford the desired product, Z-5-hexadecene.

Z-11-Hexadecen-1-ol:

Z-5-Hexadecene is then subjected to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.

Example 6. Synthesis of (Z)-11-Hexadecenol Carried Out According to Scheme 16

Z-5-Hexadecene:

Into an oven-dried three-neck RBF, N-amyl triphenylphosphnium bromide (13.98 g, 33.83 mmol) is dissolved in anhydrous toluene (30 mL). The mixture is allowed to stir via a magnetic stir bar at ambient temperature until complete dissolution of the alkyl phosphonium bromide salt is achieved. A solution of 6.57 g of potassium bis(trimethylsilyl)amide (KHMDS) in anhydrous toluene (30 mL) is then dropwise added to the reaction mixture. Upon complete addition of KHMDS solution to the reaction mixture, the reaction solution is allowed to stir for another 15 minutes, and is then cooled to −78° C. in an acetone and dry ice bath.

A solution of undecanal (4.59 mL, 22.28 mmol) in toluene (40 mL) is then drop-wise added to the reaction mixture via an addition funnel. The reaction is stirred at −78° C. for 20 minutes, then allowed to warm at room temperature with stirring for another 30 minutes. The reaction is terminated by addition of methanol (40 mL) and then concentrated under reduced pressure. The resulting residue is triturated with hexanes and white precipitate, triphenyl phosphine oxide, is removed by filtration. The process is repeated until triphenyl phosphine oxide is no longer precipitated out of the solution. The remnant triphenyl phosphine oxide is removed by passing the crude reaction product through a short bed of silica using hexane as a mobile phase. Z-5-hexadecane is obtained as a colorless oil.

Z-11-hexadecen-1-ol:

Z-5-Hexadecene is subjected to biohydroxylation according to the process disclosed in Example 1 to generate Z-11-hexadecen-1-ol. The product is isolated by extraction of the fermentation broth with organic solvent and purified by distillation.

Example 7. The Use of Mixtures of (Z)-11-Hexadecenal and (Z)-5-Hexadecenal for Pest Control of Insects with Mating Response to (Z)-11-Hexadecenal

As proof of principal that a synthetically derived pheromone composition comprised of a synthetically derived sex pheromone and a positional isomer can be used to modulate the behavior of a target insect (H. zea), the Z-5-hexadecene was subject to biohydroxylation and oxidation as described above. A mixture of Z-hexadec-11-enal and Z-hexadec-5-enal was produced as shown below.

Example 8. Wind Tunnel Studies Using Positional Isomer Z-5-Hexadecenal

Four separate experiments were conducted with the moth Helicoverpa zea and its respective pheromone components (Z-11-hexadecenal, Z-9-hexadecenal). Further, the addition of Z-5-hexadecenal, the positional isomer of the natural insect Z-11-hexadecenal pheromone, was also added to the Z-11 and Z-9 blends and tested. Upwind flight and lure location of male moths were compared for: natural ratios of pheromone (97% Z-11-hexadecenal with 3% of Z-9-hexadecenal) with and without addition of the Z-5-hexadecenal positional isomer at various ratios.

Methods

Moths (4-6 day-old males in second half of scotophase) were flown in a glass wind tunnel (120×30×30 cm). A fan pushed air into the wind tunnel at 0.4 m sec⁻¹ and a second fan exhausted air at a similar rate. To provide visual cues for navigation, the floor was covered with light colored construction paper on which small (2-5 cm) circles were drawn with marker (Experiment 1) or light colored circles that were cut from light colored construction paper were placed on the floor (Experiments 2-4). Lures were made with Soxhlet-extracted, grey-rubber septa from West. Compounds were added to the septa in 50 μL of hexane and lures were dried in a fume hood for 1 h before use. Our lures (Experiments 1 and 2) were loaded with 5 μg of the pheromone (Z-11-hexadecenal with 3% of Z-9-hexadecenal) and this treatment was compared to lures with 50, 5 or 0.5% added Z-5-hexadecenal.

Conditions in the wind tunnel (27° C., 50% relative humidity) were similar to the air in the room during bioassays and to conditions under which moths were kept before assays were run.

A moth in a release cage was held on a platform 20 cm above the floor for 15 s in the plume of pheromone and then released by turning open end of the cage toward upwind. The lure was at the same height as the moth and 90 cm upwind. Moths were given 5 min to locate the lure. Data collected were: 1) whether or not a moth contacted the pheromone lure, 2) whether or not a moth nearly contacted the lure (hovering downwind within 10 cm of the lure without contact: “close but no contact”), and 3) time until contact. Flights were recorded on video and data were collected from videos.

We ran 2 experiments:

-   -   1) 3 treatments: 2.5 μg (50%), 0.25 μg (5%), or no (0%; positive         control) Z-5-hexadecenal.     -   2) 3 treatments: 0.25 μg (5%), 0.025 μg (0.5%), or no         Z-5-hexadecenal.

Over the course of the 4 experiments, occasionally lures with no pheromone (50 uL clean hexane) were included as negative controls (n=35). Conditions in the wind tunnel were the same for all experiments.

Experiment 8.1: Natural Ratios of Pheromone (Z-11-Hexadecenal with 3% of Z-9-Hexadecenal) with and without Addition of the Z-5-Hexadecenal Positional Isomer

This experiment was performed to evaluate the response of the moths to pheromone coated lures with and without high concentrations of the Z-5-hexadecenal positional isomer

With the treatments, the moths flew upwind, although relatively few moths located the pheromone lure, and there were no significant differences between numbers of moths that flew close to the lure but did not make contact (4, 6, and 5 respectively); however, significantly more moths contacted the lure (43%) with the natural pheromone blend (0% added Z5-hexadecacenal) than with 5% added Z5-hexadecacenal (Table 15; 11%; χ₂=9.07, P<0.01) or 50% added Z11-hexadecacenal (Table 15; 14%; χ₂=8.04, P<0.01). There were no significant differences among treatments in any experiment in latency (time from the start of the bioassay and contact with the lure).

TABLE 15 Expanded Wind Tunnel Experiments with 0%, 5%, and 50% Z-5-hexadecenal # contacting Close but no time until Treatment n lure % contact contact contact (s) 0% Z5 37 16 43 4 109 5% Z5 35 4 11 6 72 50% Z5  37 5 14 5 135 Z-5 = Z-5-hexadecenal

To the surprise of the inventors, the results from Experiment 8.1 indicate that the number of Helicoverpa zea moths finding the pheromone composition that includes the Z-5-hexadecenal positional isomer was significantly reduced relative to the natural pheromone blend. The moth is unexpectedly responsive to pheromone compositions including the Z-5-hexadecenal positional isomer despite the structural difference compared to Z-11-hexadecenal as the moth species flew upwind to interact with a plume in the presence and absence of the Z-5-hexadecenal positional isomer. That is, the natural pheromone blend elicited an flew upwind flight and contact response, whereas, in the presence of the Z-5-hexadecenal positional isomer, the moths flew upwind but did not contact the lures.

Experiment 8.2 Natural Ratios of Pheromone (Z-11-Hexadecenal with 3% of Z-9-Hexadecenal) with and without Addition of the Z-5-Hexadecenal Positional Isomer

This experiment was performed to assess the response of the target moth to lower concentrations of the Z-5-hexadecenal in the natural pheromone blend.

There were no significant differences between treatments in number of moths that flew close to the lure but did not make contact (Table 16; χ₂=3.98, P>0.05). Significantly more moths contacted the lure with 0% Z5-hexadecenal (38%) than with 5% added Z5-hexadecacenal (Table 16; 17%; χ₂=4.11, P<0.05), but there was no difference in numbers contacting the lures with 0 or 0.5% Z5-hexadecacenal (38 and 44% respectively; χ₂=0.33, P>0.05).

TABLE 16 Expanded Wind Tunnel Experiments with 0%, 0.5%, and 5% Z-5-hexadecenal # contacting Close but no time until Treatment n lure % contact contact contact (s) 0% Z5 37 14 38 5 140 0.5% Z5   36 16 44 3 127 5% Z5 36 6 17 9 113 Z-5 = Z-5-hexadecenal

The results of Experiment 8.2 indicate that while normal upwind flight seems to occur when the Z-5-hexadecenal positional isomer is included in the composition, contact with the lure is reduced. Furthermore, the results indicate that the amount of the Z-5-hexadecenal positional isomer can be varied to modulate attraction and landing as more moths landed on the lure in the presence of 0% or only 0.5% Z-5 hexadecenal compared to 5% Z-5 hexadecenal, whereas more moths came close to but did not contact the lure coated with 5% Z-5 hexadecenal compared to 0% or only 0.5% Z-5 hexadecenal. Thus, the pheromone compositions described herein can be used to elicit a tunable response in a target insect by inter alia varying the ratio of the positional isomer to the natural pheromone to thereby modulate attraction and/or landing.

Conclusions from 8.1-8.2

Across all treatments, nearly all moths flew upwind and most interacted with the plume in some way. Thus, the Z-5-hexadecedenal positional isomer triggers a similar upwind flight response in male moths. In general, contact was lower for treatments with added Z-5-hexadecenal.

These results indicate that the presence of the Z-5-hexadecenal positional isomer in the natural pheromone blend reduced the number of moths contacting the lures while still maintaining upwind orientation similar to the physiological responses to the natural pheromone blend alone.

The moths therefore can respond to the Z-5-hexadecenal positional isomer, which indicates that a positional isomer has valuable applications in modulating insect behavior. Thus, the presence of the positional isomer can be used to elicit a tunable response from target insects. That is, the amount of the he Z-5-hexadecenal positional isomer in the pheromone composition can be varied to either cause the moths to fly toward the lure but not land or to land on the lure.

Some moths flew upwind when an unbaited lure was present (15 of 35) but none of these exhibited plume-oriented flight or lure contact.

Thus, a surprising and unexpected result of including the Z-5-hexadecenal positional isomer in the pheromone composition was that the number of moths finding the lures was reduced while upwind orientation was maintained relative to the natural pheromone blend (i.e., in the absence of Z-5-hexadecenal). Although the number of moths finding the lures was reduced, the ability to attract the moth species using a pheromone composition containing a positional isomer indicates that the compositions taught herein can be used to modify insect behavior. Further, because the response elicited was dependent on the relative amount of the Z-5-hexadecenal positional isomer, these results indicate the response of the targeted insects can be tuned, e.g., to attract a target insect or to cause the target insect to land, by varying the amount of the positional isomer.

Prophetic Example 9. Mixtures of (Z)-9-Tetradecenal and (Z)-5-Tetradecenal to Modulate the Behavior of Insects with Mating Response to (Z)-9-Tetradecenal

As shown below, Z-9-tetradecenal is a naturally produced sex pheromone for various lepidopteran species. Using the biohydroxylation methodology disclosed herein, a pheromone composition comprising Z-9-tetradecenal and its positional isomer Z-5-tetradecenal can be prepared as shown below.

Wind tunnel experiments will be performed as described above using pheromone compositions comprising synthetically derived sex pheromone and a positional isomer. The inventors expect the positional isomer Z-5-tetradecenal to elicit an upwind flight response.

Prophetic Example 10. Mixtures of (Z)-9-Tetradecenyl Acetate and (Z)-5-Tetradecenyl Acetate for Pest Control of Insects to Modulate the Behavior of Insects with Mating Response to (Z)-9-Tetradecenyl Acetate

The blend of sex pheromones produced by female Spodoptera frugiperda (Fall armyworm) includes an 96.4/3.6 mixture of Z-9-tetradecenyl acetate and Z-7 dodecenyl acetate.

The Z-9-tetradecenyl acetate sex pheromone produced by female Spodoptera frugiperda (Fall armyworm) is shown below. Using the biohydroxylation methodology disclosed herein, a pheromone composition comprising Z-9-tetradecenyl acetate and its positional isomer Z-5-tetradecenyl acetate can be prepared as shown below.

Wind tunnel experiments will be performed as described above using pheromone compositions comprising the synthetically derived sex pheromone and a positional isomer. The inventors expect the positional isomer Z-5-tetradecenyl acetate in a composition with Z-9-tetradecenyl acetate to elicit response physiological response.

Prophetic Example 11. Pheromone Compositions

Any of the pheromones listed in Table 1 above can be synthesized as described herein to produce a pheromone composition comprising synthetically derived mixture of a natural pheromone and a positional isomer.

Wind tunnel experiments can be performed using a pheromone composition comprising synthetically derived mixture of a natural pheromone and a positional isomer to modulate the behavior of the target insect.

Prophetic Example 12. Synthesis of a Positional Isomer with a Subterminal Functional Group

Based on the inventors' unexpected and first reported discovery that enzyme catalysts can be used to hydroxylate an unsaturated hydrocarbon substrate, thereby creating olefins with a terminal alcohol, the inventor propose using different biohydroxylation catalysts to hydroxylate carbon atoms within the carbon skeleton (i.e., subterminal carbons). A variety of P450 enzyme are known to catalyze hydroxylation of subterminal carbons to produce secondary alcohols. See, e.g., Greer et al., Plant Physiology. 2007; 143(3):653-667. It is also possible that the hydroxylase enzymes disclosed herein catalyze the formation of subterminal hydroxyl groups in low yields. Further, it is also possible to engineer an enzyme to selectively catalyze hydroxylation of an subterminal carbon.

As shown below, the hydroxyl group can be inserted on an subterminal carbon of an unsaturated hydrocarbon substrate to produce an olefinic alcohol. Subsequent oxidation, acetylation, or esterification can generate a positional isomer of a sex pheromone naturally produced by an insect species.

Based on the wind tunnel results discussed above with terminally functionalized positional isomer thereof, the inventors expect that isomers with a subterminal hydroxy group will similarly modulate the behavior of an insect.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

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Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety for all purposes. 

What we claim is:
 1. An insect pheromone composition for modifying the behavior of a target member of the order Lepidoptera, comprising: a) a first synthetically derived insect pheromone, having a chemical structure corresponding to that of a natural insect pheromone produced by a given target member of the order Lepidoptera; and b) a positional isomer of said first synthetically derived insect pheromone, wherein said positional isomer is not naturally produced by the target member of the order Lepidoptera.
 2. The insect pheromone composition of claim 1, wherein the positional isomer is not produced by a member of the order Lepidoptera.
 3. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the family Noctuidae or Plutellidae.
 4. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the genus Helicoverpa, Plutella, Spodoptera, or Chrysodeixis.
 5. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Helicoverpa zea.
 6. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Helicoverpa armigera.
 7. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Plutella xylostella.
 8. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Spodoptera frugiperda.
 9. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by Chrysodeixis includens.
 10. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone is present in the composition in a ratio of from about 99% to about 1%, relative to the positional isomer, which is present in the composition in a ratio of from about 1% to about 99%.
 11. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone is present in the composition in an amount of from about 99% to about 1% w/w.
 12. The insect pheromone composition of claim 1, wherein the positional isomer is present in the composition in an amount of from about 99% to about 1% w/w.
 13. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone is Z-11-hexadecenal.
 14. The insect pheromone composition of claim 1, wherein the first synthetically derived insect pheromone is Z-11-hexadecenal and the positional isomer is Z-5-hexadecenal.
 15. The insect pheromone composition of claim 1, further comprising: c) a second synthetically derived insect pheromone, having a chemical structure corresponding to that of a natural insect pheromone produced by a given target member of the order Lepidoptera; and d) optionally, a positional isomer of said second synthetically derived insect pheromone, wherein said positional isomer is not naturally produced by the target member of the order Lepidoptera.
 16. The insect pheromone composition of claim 15, wherein the second synthetically derived insect pheromone is Z-9-hexadecenal.
 17. The insect pheromone composition of claim 15, wherein the second synthetically derived insect pheromone is Z-9-hexadecenal and the positional isomer of the second synthetically derived insect pheromone is present and is Z-7-hexadecenal.
 18. The insect pheromone composition of claim 15, wherein the first synthetically derived insect pheromone is Z-11-hexadecenal and the positional isomer of the first synthetically derived insect pheromone is Z-5-hexadecenal, and wherein the second synthetically derived insect pheromone is Z-9-hexadecenal and the positional isomer of the second synthetically derived insect pheromone is present and is Z-7-hexadecenal.
 19. The insect pheromone composition of claim 15, further comprising: at least one additional synthetically derived insect pheromone.
 20. The insect pheromone composition of claim 15, further comprising: an agriculturally acceptable adjuvant or carrier.
 21. An insect pheromone composition for modifying the behavior of male Helicoverpa sp., comprising: a) Z-11-hexadecenal and Z-5-hexadecenal; and b) an agriculturally acceptable adjuvant or carrier.
 22. The insect pheromone composition of claim 21, wherein the Z-11-hexadecenal is present in the composition in a ratio of from about 99% to about 1%, relative to the Z-5-hexadecenal, which is present in the composition in a ratio of from about 1% to about 99%.
 23. The insect pheromone composition of claim 21, wherein the Z-11-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w and the Z-5-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w.
 24. The insect pheromone composition of claim 21, further comprising: Z-9-hexadecenal.
 25. The insect pheromone composition of claim 21, further comprising: Z-9-hexadecenal and Z-7-hexadecenal.
 26. The insect pheromone composition of claim 25, wherein the Z-11-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w, the Z-5-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w, the Z-9-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w, and the Z-7-hexadecenal is present in the composition in an amount of from about 99% to about 1% w/w.
 27. A method of attracting an adult male Helicoverpa sp. to a locus, comprising: presenting an effective amount of the insect pheromone composition of claim 21 to a locus.
 28. A method of attracting and killing an adult male Helicoverpa sp., comprising: presenting an effective amount of the insect pheromone composition of claim 21 to a locus, wherein said locus also comprises a mechanism to kill the Helicoverpa sp.
 29. A method of suppressing a population of Helicoverpa sp. in a given area, comprising: applying an effective amount of the insect pheromone composition of claim 21 to a locus within said area.
 30. A method of suppressing a population of Helicoverpa sp. in a given area, comprising: permeating the atmosphere within said area with an effective amount of the insect pheromone composition of claim
 21. 31. The method of suppressing a population of Helicoverpa sp. in a given area, according to claim 29, wherein the effective amount of the insect pheromone composition is sufficient to at least partially disrupt mating within the Helicoverpa sp. population.
 32. The method of suppressing a population of Helicoverpa sp. in a given area, according to claim 30 wherein the effective amount of the insect pheromone composition is sufficient to at least partially disrupt mating within the Helicoverpa sp. population.
 33. An insect pheromone composition for modifying the behavior of a target insect, comprising: a) a first synthetically derived insect pheromone having a chemical structure corresponding to the chemical structure of a naturally occurring insect pheromone produced by the target insect, said structure comprising formula (1),

wherein R is located on a terminal carbon of an m-end of a carbon-carbon double bond in an unsaturated hydrocarbon substrate; and b) a positional isomer of said first synthetically derived insect pheromone, said positional isomer having a chemical structure of formula (2),

wherein the positional isomer has an R located on a terminal carbon of an v-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; wherein m and n are independently integers from 0 to 15, wherein a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, ore is 1, wherein b and d are independently integers from 0 to 10, and the m-end and the n-end are located on opposing sides of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; and wherein each R is independently —OH, ═O, or —OAc.
 34. The insect pheromone composition of claim 33, further comprising an analog of said first synthetically derived insect pheromone, said analog having a chemical structure of formula (3)

wherein the analog has an R′ located on the n-end and the m-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; wherein m and n are independently integers from 0 to 15, wherein a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, ore is 1, wherein b and d are independently integers from 0 to 10, and the m-end and the n-end are located on opposing sides of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; and wherein each R′ is independently H, —OH, ═O, —OAc, or —OOH.
 35. The insect pheromone composition of claim 33, further comprising: a positional isomer of said first synthetically derived insect pheromone, said positional isomer having a chemical structure of formula (4),

wherein the positional isomer has an R located on a subterminal carbon on the m-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate thereby forming an i-end, wherein the i-end comprises a terminal carbon of the unsaturated hydrocarbon substrate; or a positional isomer of said first synthetically derived insect pheromone, said positional isomer having a chemical structure of formula (5),

wherein the positional isomer has an R located on a subterminal carbon on the n-end of the carbon-carbon double bond in the unsaturated hydrocarbon substrate, thereby forming an i-end, wherein the i-end comprises a terminal carbon of the unsaturated hydrocarbon substrate; wherein m, n, and i are independently integers from 0 to 15, wherein a, c, and e are independently integers from 0 to 1, provided that at least one of a, c, ore is 1, wherein b and d are independently integers from 0 to 10 and the m-end and the n-end are located on opposing sides of the carbon-carbon double bond in the unsaturated hydrocarbon substrate; and wherein R is —OH, ═O, or —OAc.
 36. The insect pheromone composition of 33, wherein the sum of a, b, c, d, e, m, and n is an integer from 6 to
 20. 37. The insect pheromone composition of 34, wherein the sum of a, b, c, d, e, m, and n is an integer from 6 to
 20. 38. The insect pheromone composition of 35, wherein the sum of a, b, c, d, e, i, m, and n is an integer from 6 to
 20. 39. The insect pheromone composition of claim 33, wherein the first synthetically derived pheromone has the following chemical structure: a)


40. The insect pheromone composition of claim 33, wherein the first synthetically derived insect pheromone has the following chemical structure: a)

and wherein the positional isomer has the following chemical structure:


41. The insect pheromone composition of 33, further comprising a second synthetically derived insect pheromone having a chemical structure corresponding to the chemical structure of a naturally occurring insect pheromone produced by the target insect, wherein said second synthetically derived insect pheromone has the following chemical structure: c)

and d) optionally, a positional isomer of said second synthetically derived insect pheromone, wherein said positional isomer is not naturally produced by the target insect.
 42. The insect pheromone composition of claim 41, wherein the positional isomer is present and has the following chemical structure:


43. The insect pheromone composition of claim 41, wherein the first synthetically derived insect pheromone has the following chemical structure: a)

and the positional isomer of the first synthetically derived insect pheromone has the following chemical structure: b)

and wherein the second synthetically derived insect pheromone has the following chemical structure c)

and the positional isomer of the second synthetically derived insect pheromone is present and has the following chemical structure: d)


44. The insect pheromone composition of claim 33, wherein the positional isomer is not produced by the target insect.
 45. The insect pheromone composition of claim 33, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the order Lepidoptera.
 46. The insect pheromone composition of claim 33, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the family Noctuidae or Plutellidae.
 47. The insect pheromone composition of claim 33, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by a member of the genus Helicoverpa, Plutella, Spodoptera, or Chrysodeixis.
 48. The insect pheromone composition of claim 33, wherein the first synthetically derived insect pheromone has a chemical structure corresponding to that of a natural insect sex pheromone produced by an insect selected from the group consisting of Helicoverpa zea, Helicoverpa armigera, Plutella xylostella, Spodoptera frugiperda, and Chrysodeixis includens. 